Phosphatases which activate map kinase pathways

ABSTRACT

The present invention provides novel JNK activating phosphatase polypeptides and nucleic acid molecules encoding the same. The invention also provides vectors, host cells, antibodies and methods for producing JNK activating phosphatase polypeptides. Also provided for are methods for the diagnosis and treatment of diseases associated with JNK activating phosphatase polypeptides.

FIELD OF THE INVENTION

The present invention relates to novel JNK activating phosphatasepolypeptides and nucleic acid molecules encoding the same. The inventionalso relates to vectors, host cells, antibodies and methods forproducing JNK activating phosphatase polypeptides. Also provided for aremethods for the diagnosis and treatment of diseases associated with JNKactivating phosphatase polypeptides.

BACKGROUND OF THE INVENTION

Technical advances in the identification, cloning, expression, andmanipulation of nucleic acid molecules have greatly accelerated thediscovery of novel therapeutics based upon deciphering the human genome.Rapid nucleic acid sequencing techniques can now generate sequenceinformation at unprecedented rates and, coupled with computationalanalyses, allow the assembly of overlapping sequences into entiregenomes and the identification of polypeptide-encoding regions.Comparison of a predicted amino acid sequence against a databasecompilation of known amino acid sequences can allow one to determine theextent of homology to previously identified sequences and/or structurallandmarks. Cloning and expression of a polypeptide-encoding region of anucleic acid molecule provides a polyp eptide product for structural andfunctional analysis. Manipulation of nucleic acid molecule and encodedpolypeptides to give variants and derivatives thereof may conferadvantageous properties on a product for use as a therapeutic.

In spite of the significant technical advances in genome research overthe past decade, the potential for development of novel therapeuticsbased on the human genome is still largely unrealized. Genes encodingpotentially beneficial protein therapeutics, or those encodingpolypeptides that may act as “targets” for therapeutic molecules havestill not been identified. In addition, structural and functionalanalyses of polypeptide products from many human genes have not beenundertaken.

Accordingly, it is an object of the invention to identify novelpolypeptides and nucleic acid molecules encoding the same which havediagnostic or therapeutic benefit.

SUMMARY OF THE INVENTION

The present invention relates to novel JNK activating phosphatasenucleic acid molecules and encoded polypeptides.

The invention provides for an isolated nucleic acid molecule comprisinga nucleotide sequence selected from the group consisting of:

-   -   (a) the nucleotide sequence as set forth in SEQ ID NOS: 1 or 3;    -   (b) a nucleotide sequence encoding the polypeptide as set forth        in SEQ ID NOS: 2 or 4;    -   (c) a nucleotide sequence corresponding to nucleotide position        number 181 to 795 in SEQ ID NO: 1 or nucleotide position number        15 to 629 in SEQ ID NO:3;    -   (d) a nucleotide sequence encoding a polypeptide that is at        least about 80, 85, 90, 95, 96, 97, 98, or 99 percent identical        to the nucleotide sequence of (c);    -   (e) an allelic variant or splice variant of any of (a), (b, (c)        or (d);    -   (f) a nucleotide sequence of (b), (c), (d) or (e) encoding a        polypeptide fragment of at least about 25, 50, 75, 100, or        greater than 100 amino acid residues wherein the polypeptide        fragment has an activity of regulating JNK activation or        modulating JNX-mediated signal transduction;    -   (g) a nucleotide sequence of (a), (b), (c) or (d) comprising a        fragment of at least about 10, 15, 20, 25, 50, 75, 100, or        greater than 100 nucleotides;    -   (h) a nucleotide sequence encoding a polypeptide that has a        substitution and/or deletion of 1 to 100 amino acid residues as        set forth in any of SEQ ID NOS: 2 or 4 wherein the polypeptide        has an activity of regulating JNK activation or modulating        JNK-mediated signal transduction; or serves as an antigen for        generating antibodies; and    -   (i) a nucleotide sequence which hybridizes under stringent        conditions to any of (a)-(h);    -   (j) a nucleotide sequence complementary to any of (a)-(h).

The invention also provides for an isolated polypeptide comprising theamino acid sequence selected from the group consisting of:

-   -   (a) the amino acid sequence as set forth in SEQ ID NOS: 2 or 4;    -   (b) a fragment of the amino acid sequence set forth in SEQ ID        NOS: 2 OR 4 comprising at least about 25, 50, 75, 100, or        greater than 100 amino acid residues wherein the fragment has an        activity of regulating JNK activation or modulating JNK-mediated        signal transduction; or serves as an antigen for generating        antibodies;    -   (c) an ortholog of SEQ ID NOS: 2 or 4; and    -   (d) an allelic variant or splice variant of (a) or (c).

The invention also provides for an expression vector comprising thenucleic acid molecules as set forth above, host cells comprising theexpression vectors of the invention, and a method of production of anJNK activating phosphatase polypeptide comprising culturing the hostcells and optionally isolating the polypeptide so produced.

A transgenic non-human animal comprising a nucleic acid moleculeencoding an JNK activating phosphatase polypeptide is also encompassedby the invention. The JNK activating phosphatase nucleic acid moleculesare introduced into the animal in a manner that allows expression andincreased levels of an INK activating phosphatase polypeptide, which mayinclude increased circulating levels. Alternatively, the JNK activatingphosphatase nucleic acid molecules are introduced into the animal in amanner that prevents expression of endogenous JNK activatingphosphastase (i.e., generates a transgenic animal possessing an JNKactivating phosphatase gene knockout). The transgenic non-human animalis preferably a mammal, and more preferably a rodent, such as a rat or amouse.

Also provided are derivatives of the JNK activating phosphatasepolypeptides of the invention, fusion polypeptides comprising the JNKactivating phosphatase polypeptides of the invention, and antibodiesspecifically binding the JNK activating phosphatase polypeptides of theinvention.

Compositions comprising the nucleotides or polypeptides of the inventionand a carrier, adjuvant, solubilizer, stabilizer or anti-oxidant, orother pharmaceutically acceptable agent are also encompassed by theinvention. The compositions may include pharmaceutical compositionscomprising therapeutically effective amounts of the nucleotides orpolypeptides of the invention, and methods of using the polypeptides andnucleic acid molecules.

Surprisingly, JNK activating phosphatase polypeptide was differentiallyexpressed in hematopoietic cells, thereby distinguishing it from otherdual-activity phosphatases. Therefore, the present polypeptide, and itsuseful nucleic acid intermediates, may have utility in differentiatinghematopoietic cells from background.

The JNK activating phosphatase polypeptides and nucleic acid moleculesof the invention may be used to screen for therapeutic agents to treat,prevent and/or detect conditions relating to JNK-mediated disorders. Theinvention provides for treating, preventing or ameliorating aJNK-mediated disorder comprising administering to an animal an INKactivating phosphatase polypeptide. The invention also provides for amethod of diagnosing a JNK-mediated disorder or a susceptibility to aJNK-mediated disorder in an animal which includes both determining thepresence or amount of expression of an JNK activating phosphatasepolypeptide and diagnosing a JNK-mediated disorder or a susceptibilityto a JNK-mediated disorder based on the presence or amount of expressionof an JNK activating phosphatase polypeptide. The animal is preferably amammal, and more preferably a human. The present invention also relatesto methods for manufacture of a medicament for the treatment ofJNK-mediated disorders.

In mammalian cells, a specific protein kinase pathway is activated inresponse to stress due to, for instance, inflammatory cytokines,chemotherapeutic drugs and UV or ionizing radiation. The term“JNK-mediated disorder” is used to describe a pathological conditionresulting, at least in part, from excessive activation of a c-Junamino-terminal kinase (JNK) signal transduction pathway in response tosuch a stress. JNK-mediated disorders may include, for example, ischemicheart disease, cardiac hypertrophy, burns due to heat or radiation (UV,X-ray, gamma, beta, etc.), kidney failure, liver damage due to oxidativestress or alcohol, respiratory distress syndrome, septic shock,rheumatoid arthritis, autoimmune disorders, and other types ofinflammatory diseases. JNK-mediated disorders further includeproliferative disorders. Examples of stress-related JNK-mediatedproliferative disorders might include psoriasis and malignancies ofvarious tissues of the body, such as malignancies of the skin, bonemarrow, lung, liver, breast, gastrointestinal system, and genito-urinarytract The present invention provides a polypeptide (a phosphatase)involved in the JNK signal transduction pathway. The polypeptidemodulates the response of a c-Jun amino-terminal kinase to cytokines orother stimuli, thereby regulating JNK activation and modulatingJNK-mediated signal transduction. The modulation of JNK activityincludes inhibitory or stimulatory effects. In some cases, augmentationof JNK activity is desirable, e.g., induction of apoptosis.

A therapeutic agent that inhibits JNK activity will interfere with theJNK-mediated signal transduction pathway. For example, a therapeuticagent may alter the protein kinase activity of JNK, decrease the levelof JNK transcription or translation (e.g., an antisense polynucleotideable to bind JNK mRNA), or alter JNK phosphorylation, thus disruptingthe JNK-mediated signal transduction pathway. Further examples of suchagents include antibodies that bind specifically to JNK-activatingpolypeptides, and fragments of JNK-activating polypeptides thatcompetitively inhibit JNK activity.

A therapeutic agent that promotes or enhances JNK activity supplements aJNK-mediated signal transduction pathway. For example, a promoting agentcan be administered in instances where the JNK-mediated disorder iscaused by under-expression of the JNK-activating polypeptide. Inaddition, portions of DNA encoding a JNK-activating polypeptide can beintroduced into cells that under-express a JNK-activating polypeptide.

The polypeptides of the present invention may be used to screentherapeutic agents for treating a disease involving cytokine productionin an animal. The agent may regulate cellular response toproinflammatory cytokines produced as a result of exposure to certainstressors. Such diseases include medical disorders and diseases in whichthe pathogenesis of the disease and/or the physiological effects of thedisease might be ameliorated by regulation of response toproinflammatory cytokines. Such diseases include, but are not limitedto, allergic diseases, anaphylaxis, inflammation, mast cell disorders,sepsis and cancer.

The polypeptides and polynucleotides of the invention are useful foridentifying agents which modulate the JNK signal transduction pathways.Agents that inhibit a JNK signal transduction pathway can be used in thetreatment of JNK-mediated disorders, as described above. Agents thatstimulate a JNK signal transduction pathway can be used in a number ofways, including inducing programmed cell death (apoptosis) in tissues.For example, the elimination of UV damaged cells can be used to preventcancer.

The present invention further provides the means for both acute andprophylactic treatment of stress-related and inflammatory disorders. Forexample, it is envisioned that ischemic heart disease may be treatedduring episodes of ischemia and oxidative stress following reperfusion.In addition, a patient at risk for ischemia may be treated prior toischemic episodes. In another example, a therapeutic agent whichinhibits JNK activity may be administered to control inflammatoryresponses by inhibiting the response to inflammatory cytokines,including TNF and IL-1. Stress-related proliferative disorders can alsobe treated by administering a therapeutic agent that inhibits JNKactivity. Such therapeutic agents can be used alone or in combinationwith other therapeutic agents, for example, with chemotherapeutic agentsin the treatment of malignancies.

Agents used to modulate JNK activity, include polynucleotides,polypeptides, and other molecules such as antisense oligonucleotides andribozymes. Thus, the present invention provides means for treating aJNK-mediated disorders by administering to a subject in need thereof aneffective dose of a therapeutic agent that modulates (inhibits orenhances, as required) the activity of JNK As used herein, the term“therapeutic agent” means any compound or molecule that achieves thedesired effect on a JNK-mediated disorder when administered to a subjectin need thereof Therapeutic uses of such agents include but are notlimited to the following: acute pancreatitis; ALS; Alzheimer's disease;cachexia/anorexia; asthma; atherosclerosis; chronic fatigue syndrome,fever, diabetes (e.g., insulin diabetes); glomerulonephritis; graftversus host rejection; hemohorragic shock; hyperalgesia, inflammatorybowel disease; inflammatory conditions of a joint, includingosteoarthritis, psoriatic arthritis and rheumatoid arthritis; ischemicinjury, including cerebral ischemia (e.g., brain injury as a result oftrauma, epilepsy, hemorrhage or stroke, each of which may lead toneurodegeneration); lung diseases (e.g., ARDS); multiple myeloma;multiple sclerosis; myelogenous (e.g., AML and CML) and other leukemias;myopathies (e.g., muscle protein metabolism, especially in sepsis);osteoporosis; Parkinson's disease; pain; pre-term labor, psoriasis;reperfusion injury; septic shock; side effects from radiation therapy,temporal mandibular joint disease, tumor metastasis; or an inflammatorycondition resulting from strain, sprain, cartilage damage, trauma,orthopedic surgery, infection or other disease processes.

JNK- or JNK-like activity also appears to be involved in cardiachypertrophy. See for example, Silberbach et al., J. Biol. Chem., 274(35):24858-24864 (1999); Yano et al., Circ. Res., 83 (7): 752-760(1998); Adams et al., Circ.

Res., 83 (2):167-178 (1998); Memoto et al., Mol. Cell. Biol., 18 (6):3518-3526 (1998); Wang et al., J. Biol. Chem., 273 (10): 5423-5426(1998); and Ramirez et al., J. Biol. Chem., 272 (22):14057-14061 (1997).An inhibitor of JNK activity may ameliorate such an event.

The present invention provides a means to produce agents useful in theprophylaxis and treatment of tumor necrosis factor a (TNF-α) mediatedand interleukin-1 (IL- 1) mediated diseases as well as other maladies,such as diseases involving inflammation, pain and diabetes, through theinhibition or reduction of JNK activity. JNK appears to be activated byTNF-α and IL-1 and appears to be involved in their signaling pathway.Inhibition or reduction of JNK activity could mitigate the effects ofTNF-α, IL-1β, IL-6 and/or IL-8, in particular elevated levels of TNF-αand IL-1. Thus, the inhibition or reduction of JKAP activity wouldinhibit or reduce JNK activity and thereby mitigate the effects of thissignaling pathway.

IL-1 and TNF-α are pro-inflammatory cytokines secreted by a variety ofcells, including monocytes and macrophages, in response to manyinflammatory stimuli (e.g., lipopolysaccharide-LPS) or external cellularstress (e.g., osmotic shock and peroxide). Elevated levels of TNF-αand/or IL-1 over basal levels have been implicated in mediating orexacerbating a number of disease states including rheumatoid arthritis;Pagets disease; osteoporosis; multiple myeloma; uveititis; acute andchronic myelogenous leukemia; pancreatic β cell destruction;osteoarthritis; rheumatoid spondylitis; gouty arthritis; inflammatorybowel disease; adult respiratory distress syndrome (ARDS); psoriasis;Crohn's disease; allergic rhinitis; ulcerative colitis; anaphylaxis;contact dermatitis; asthma; muscle degeneration; cachexia; Reiter'ssyndrome; type I and type II diabetes; bone resorption diseases; graftvs. host reaction; ischemia reperfusion injury; atherosclerosis; braintrauma; multiple sclerosis; cerebral malaria; sepsis; septic shock;toxic shock syndrome; fever, and myalgias due to infection. HIV-1,HIV-2, HIV-3, cytomegalovirus (CNM), influenza, adenovirus, the herpesviruses (including HSV-1, HSV-2), and herpes zoster are also exacerbatedby TNF-α.

Also, TNF-α has been reported to play a role in head trauma, stroke, andischemia (Shohami et al., J. Cereb. Blood Flow Metab. 14, 615 (1994);Feurstein et al., Neurosci. Lett.. 164, 125 (1993)). Administration ofTNF-α into the rat cortex has been reported to result in significantneutrophil accumulation in capillaries and adherence in small bloodvessels. TNF-α promotes the infiltration of other cytokines (IL-1β,IL-6) and also chemokines, which promote neutrophil infiltration intothe infarct area (Feurstein, Stroke 25, 1481 (1994)). TNF-α haas alsobeen implicated to play a role in type II diabetes (Endocrinol.130,43-52, 1994; and Endocrinol. 136, 1474-1481, 1995). TNF-α alsoappears to play a role in promoting certain viral life cycles anddisease states associated with them (Clouse et al., J. Immunol. 142,431(1989); Lahdevirta et al., Am. J. Med. 85, 289 (1988)).

TNF-α is upstream in the cytokine cascade of inflammation. As a result,elevated levels of TNF-α may lead to elevated levels of otherinflammatory and proinflammatory cytokines, such as IL-1, IL-6, andIL-8. Elevated levels of IL-1 over basal levels have been implicated inmediating or exacerbating a number of disease states includingrheumatoid arthritis; osteoarthritis; rheumatoid spondylitis; goutyarthritis; inflammatory bowel disease; adult respiratory distresssyndrome (ARDS); psoriasis; Crohn's disease; ulcerative colitis;anaphylaxis; muscle degeneration; cachexia; Reiter's syndrome; type Iand type II diabetes; bone resorption diseases; ischemia reperfusioninjury; atherosclerosis; brain trauma; multiple sclerosis; sepsis;septic shock; and toxic shock syndrome. Viruses sensitive to TNF-αinhibition, e.g., HIV-1, HIV-2, HIV-3, are also affected by IL-1. IL-1also appears to play a role in promoting certain viral life cycles(Folks et al., J. Immunol. 136, 40 (1986)). Beutler et al. (J. Immunol.135, 3969 (1985)) discussed the role of IL-1 in cachexia. Baracos et al.(New Eng. J. Med. 308, 553 (1983)) discussed the role of IL-1 in muscledegeneration. TNF-α and IL-1 appear to play a role in pancreatic β celldestruction and diabetes. Pancreatic β cells produce insulin which helpsmediate blood glucose homeostasis. Deterioration of pancreatic β cellsoften accompanies type I diabetes. Pancreatic β cell functionalabnormalities may occur in patients with type II diabetes. Type IIdiabetes is characterized by a functional resistance to insulin.Further, type II diabetes is also often accompanied by elevated levelsof plasma glucagon and increased rates of hepatic glucose production.Glucagon is a regulatory hormone that attenuates liver gluconeogenesisinhibition by insulin. Glucagon receptors have been found in the liver,kidney and adipose tissue. Thus glucagon antagonists are useful forattenuating plasma glucose levels (WO 97/16442, incorporated herein byreference in its entirety). By antagonizing the glucagon receptors, itis thought that insulin responsiveness in the liver will improve,thereby decreasing gluconeogenesis and lowering the rate of hepaticglucose production.

In rheumatoid arthritis, both IL-1 and TNF-α induce synoviocytes andchondrocytes to produce collage ase and neutral proteases, which leadsto tissue destruction within the arthritic joints. In rheumatoidarthritis models in animals, multiple intra-articular injections ofTNF-α or IL-1 have led to an acute and destructive form of arthritis(Brahn et al., Lymphokine Cytokine Res. 11, 253 (1992); and Cooper,Clin. Exp. Inmunol. 898, 244 (1992); Chandrasekhar et al., ClinicalImmunol Immunopathol. 55, 382 (1990)). In studies using culturedrheumatoid synovial cells, IL-1 is a more potent inducer of stromelysinthan is TNF-α (Firestein, Am. J. Pathol 140, 1309 (1992)). At sites oflocal injection, neutrophil, lymphocyte, and monocyte emigration hasbeen observed, which is attributed to the induction of chemokines (e.g.,IL-8), and the up-regulation of adhesion molecules (Dinarello, Eur.Cytokine Netw. 5, 517-531 (1994)).

The invention also provides for a method of identifying a test moleculewhich binds to an JNK activating phosphatase polypeptide wherein themethod comprises contacting an JNK activating phosphatase polypeptidewith a test molecule and determining the extent of binding of the testmolecule to the polypeptide. The method further comprises determiningwhether such test molecules are agonists or antagonists of an JNKactivating phosphatase polypeptide.

The invention also provides for a method of testing the impact ofmolecules on the expression of JNK activating phosphatase polypeptide oron the activity of JNK activating phosphatase polypeptide.

A method of regulating expression and modulating (i.e., increasing ordecreasing) levels of an JNK activating phosphatase polypeptide are alsoencompassed by the invention. One method comprises administering to ananimal a nucleic acid molecule encoding an JNK activating phosphatasepolypeptide. In another method, a nucleic acid molecule comprisingelements that regulate expression of an JNK activating phosphatasepolypeptide may be administered. Examples of these methods include genetherapy and anti-sense therapy.

DESCRIPTION OF THE FIGURES

The foregoing and other aspects and advantages of the invention will beapparent on consideration of the following detailed description and theaccompanying drawings, wherein:

FIG. 1 illustrates the nucleotide sequence of the human JNK activatingphosphatase gene (SEQ ID NO: 1) and the deduced amino acid sequence ofhuman JNK activating phosphatase protein (SEQ ID NO: 2).

FIG. 2 illustrates the nucleotide sequence of the mouse JNK activatingphosphatase gene (SEQ ID NO: 3) and the deduced amino acid sequence ofmouse JNK activating phosphatase protein (SEQ ID NO: 4).

FIG. 3 illustrates the JKAP amino acid sequence analysis and expressionpattern. (A) Amino acids sequences corresponding to the catalyticdomains of selected MAPK phosphatases were aligned in PIMA 1.4 usingsequential branching clustering. The resulting alignment was processedin BOXSHADE. Sequence identities are highlighted in black; similaritiesare highlighted in gray. Gaps are indicated by dots. The alignedsequences include mJKAP (SEQ ID NO: 5); puckered (SEQ ID NO: 6); rMKP-3(SEQ ID NO: 7); rMKP-X (SEQ ID NO: 8); hMKP-4 (SEQ ID NO: 9); rMKP-2(SEQ ID NO: 10); mNWP-1 (SEQ ID NO: 11); mH3/6 (SEQ ID NO: 12); mPAC-1(SEQ ID NO: 13); hVH3 (SEQ ID NO: 14); and hVHR (SEQ ID NO: 15) and theresulting consensus sequence (SEQ ID NO: 16). (B) Phylogenetic analysiswas carried out on aligned sequences by parsimony in PHYLIP usingPROTPARS with bootstrapping of 1000 replicates. The numbers correspondto the occurrences of the branch in the consensus tree. (C)Polyadenylated mRNA from adult mouse tissues was hybridized with a JKAPcDNA probe. mRNA integrity and quantity was confirmed by hybridizationwith β-actin. Molecular weights in kilobase pairs are indicated on theleft.

FIG. 4 illustrates the In situ expression analysis of JKAP. (A and B)Lin⁻ Sca-1⁺ cells (A) and Lin⁻ Sca-1⁻ cells (B) obtained from adultmouse bone marrow by FACS were hybridized with a JKAP cDNA fragment (Cthrough E) E10.5 mouse embryos were hybridized with a JKAP antisenseriboprobe, 20X (C) and a JKAP sense riboprobe, 20X (D). The caudalregion is shown at 50X (E). (F) 10 μm transverse sections were takenthrough the caudal region of the embryo in (C), 200X (JWB).

FIG. 5 illustrates activation of JNK, but not p38 or ERK, by JKAP intransfected 293T cells. (A) 293T cells (1.5×10⁵ cells/35 mm well) weretransfected with 0.1 μg of HA-JNK alone, HA-JNK plus 2 μg of either JKAPor JKAP-C88S. (B) 293T cells (1.5×10⁵ cells/35 mm well) wereco-transfected with 2 μg of HA-p38 and various amounts of JKAP. As acontrol, co-transfection of HA-p38 and 2 μg of MKK6 was included. (C)293T cells (1.5×10⁵ cells/35 mm well) were co-transfected with 2 μg ofHA-ERK2 and various amounts of JKAP. As a control, co-transfection ofHA-ERK 2 and 0.5 μg of PKC-ξ was included. Empty vectors were used tonormalize the amount of transfected DNA. At 44 h post-transfection,cells were collected and cell lysates prepared. HA-JNK1, HA-p38, andHA-ERK2 were immunoprecipitated with an anti-HA antibody (12CA5), andimmunocomplex kinase assays were performed using GST-cJun (1-79),GST-ATF2, and MBP as substrates, respectively. Equivalent levels ofHA-JNK, HA-p38, and HA-ERK2 expression were verified by immunoblotanalysis using anti-HA (12CA5). (D) Blocking of TNF-alpha induced JNKactivity by mutant JKAP. 293T cells transfected with 0.1 ug of HA-JNK1alone or HA-JNK1 plus 2 ug of JECAP88S were treated with TNF-alpha (10ng/ml). After 30 minutes, cells were collected and cell lysatesprepared. HA-JNK1 was immunoprecipitated with an anti-HA antibody(12CA5), and immunocomplex kinase assays were performed using GST-cJun(1-79) as a substrate. Equivalent levels of HA-JNK1 expression wereverified by immunoblot (DB) analysis using anti-HA (12CA5).

FIG. 6 illustrates the generation of JKAP^(−/−)ES cells. (A) The JKAPgenomic locus, targeting vector, and mutated locus are schematicallyrepresented. Restriction enzyme sites (B. BamHI; X, XbaI; S, SalI) andthe probe used to detect targeting events are indicated. (B) Genomic DNAwas isolated from JKAP^(+/+) and JKAP^(−/−), and JKAP^(−/−)ES cells,which were derived through selection of JKAP^(±)ES cell lines in 2 mg/nLG418. The DNA was digested with BamHI, transferred for Southernanalysis, and hybridized with a probe flanking the 5′ insertion site ofthe targeting vector. Molecular weights in kilobase pairs are indicatedon the left (C) Total RNA from JKAP^(+/+), JKAP^(±), and JKAP^(−/−)EScells was isolated and hybridized with a JKAP cDNA probe. RNA integrityand quantity was evaluated by methylene blue staining after Northertransfer. Molecular weight in kilobase pairs is indicated on the left.

FIG. 7 illustrates the reduced response of JKAP^(−/−)ES cells to respondto (A) TNF-α, (B) TFG-β, (C) Il-1 and (D) sorbitol, but not (E) UV-C,for JNK activation. ˜80% confluence of JKAP^(+/+) and JKAP^(−/−)ES cellswere treated with TNF-α (10 ng/mL), TGF-β (10 ng/mL), and IL-1 (10ng/mL) for 10 min and sorbitol (400 mM), and UV-C (300 J/m²) for 30 minat 37° C. Cells were then collected and cell lysates prepared.Endogenous JNK1 was immunoprecipitated with an anti-JNK1 Antibody(Ab101), and immunocomplex kinase assays were performed using GST-cJun(1-79) as a substrate. (F) The expression levels of JNK1 JKAP^(+/+),JKAP^(±), and JKAP^(−/−)ES cells were monitored by immunoblot analysisusing anti-JNK1 antibody (Ab101). (G) Cultures of fibroblasts derivedfrom JKAP +/+and JKAP -/- embryos were stimulated with 100 ng/ml4alpha-phorbol 12-myristate 13-acetate (PMA). After various times celllysates were prepared and immunoprecipitated with anti-ERK2. ERK2 kinaseactivity was measured by phosphorylation of MBP. MBP was separated onPAGE gel and the phosphorylated product measured by phsphorimageranalysis. (H) Cultures of fibroblasts derived from JKAP +/+ and JKAP −/−embryos were stimulated with UV-C (100J/m2) and at various timesthereafter, immunocomplex assays for p38 activity were performed.Endogenous p38 was immunoprecipitated and then its kinase activity wasmeasured on either MBP or GST-ATF2 with similar results.

FIG. 8 illustates the co-expression of Myc-tagged JKAP with HPK-1 in293T cells. (A) Lysates were prepared and HPK-1 was immunoprecipitatedwith an anti-HPK-1 antibody (484). Co-immunoprecipitated JKAP wasdetected with a commercially available anti-myc antibody. Equivalentlevels of HPK-1 expression were confirmed by immunoblot analysis using acommercially available anti-HPK-1 antibody (484). (B) 293T cells wereco-transfected with 0.1 ug of HA-JNK1 alone, HA-JNK1 plus 2 ug of eitherJKAP or HPK-1, or HA-JNK1 plus 2 ug each of both JKAP and HPK-1. SeeFIG. 8(b). Empty vectors were used to normalize the amount oftransfected DNA. Cell lysates were prepared, and HA-JNK1 wasimmunoprecipitated with an anti-HA antibody (12CA5). Immunocomplexkinase assays were performed using GST-cJune (1-79) as a substrate(WESTERN). (C) 292T cells were transfected with 2 ug empty vectorcontrol, myc-JKAP, or myc-JKAP-C88S. Cell lysates were prepared andimmunoprecipitated with a commercially available anti-myc antibody. Theresulting precipitates were assayed for phosphatease activity byhydrolysis of p-ntitrophenyl phosphate (pNPP). The enzyme reaction wasterminated after 30 minutes by addition of 3N NaOH and the productmeasured spectrophotometrically at 410 nm.

DETAILED DESCRIPTION OF THE INVENTION

The section headings herein are for organizational purposes only and arenot to be construed as limiting the subject matter described therein.All references cited in this application are expressly incorporated byreference herein.

A novel dual-specificity phosphatase, a JNK activating phosphatase(JKAP) that selectively upregulates the JNK pathway was cloned andcharacterized. This protein appears to be required for maximal JNKactivation.

Overexpression of JNK activating phosphatase specifically activated theJNK cascade. Targeted gene disruption in cultured embryonic stem cellsdemonstrated that JNK activating phosphatase was necessary for inductionof JNK activity by proinflammatory cytokines and osmotic stress, but notultraviolet (UV) irradiation. These data suggest a new role forphosphatases in the regulation of signaling by MAP kinases.

Much interest in phosphatases has stemmed from the concept thatphosphatases serve to “turn off” what kinases “turn on”. Certainly,previously identified dual-specificity phosphatases have primarilyinactivated MAPK's, through dephosphorylation of the threonine andtyrosine residues in the TXY motif of the catalytic domain of the kinase(M. H. Cobb and E. J. Goldsmith, J. Biol. Chem. 270, 14843 (1995)). JNKactivating phosphatase uniquely activates the JNK pathway, but through amechanism which remains to be resolved. Direct dephosphorylation ofinhibitory residues on JNK is one possibility, as is inactivation of aninhibitory protein. JNK activating phosphatase may activate a kinasecomponent upstream of JNK, rather than JNK directly.

The mechanism through which JNK activating phosphatase exhibits pathwayspecificity for JNK over ERK and p38 also remains unclear. Other MAPkinase phosphatases are predicted to determine pathway specificitythrough a non-catalytic N-terminal domain, which is responsible forsubstrate binding (M. Camps et al., Science 280, 1262 (1998); M. Muda etal., J. Biol. Chem. 273, 9323 (1998)). JNK activating phosphataseretains high specificity for JNK despite the lack of an N-terminaldomain. The specificity of JNK activating phosphatase could be inherentin the catalytic pocket, or perhaps an interacting protein providessubstrate selectivity.

The requirement for JNK activating phosphatase in stimulus-induced JNKactivation is a novel observation among MAP kinase phosphatases. This,along with the other unusual characteristics of JNK activatingphosphatase, supports a more complex role of phosphatases in MAP kinasesignaling than as a simple “off switch”.

Definitions

The term “JNK activating phosphatase nucleic acid molecule” refers to anucleic acid molecule comprising or consisting essentially of anucleotide sequence as set forth in SEQ ID NOS: 1 or 3, comprising orconsisting essentially of a nucleotide sequence encoding the polypeptideas set forth in SEQ ID NOS: 1 or 3.

Related nucleic acid molecules comprise or consist essentially of anucleotide sequence that is about 70 percent identical to the nucleotidesequence as shown in SEQ ID NOS: 1 or 3, or comprise or consistessentially of a nucleotide sequence encoding a polypeptide that isabout 70 percent identical to the polypeptide as set forth in SEQ IDNOS: 2 or 4. In preferred embodiments, the nucleotide sequences areabout 75 percent, or about 80 percent, or about 85 percent, or about 90percent, or about 95, 96, 97, 98, or 99 percent identical to thenucleotide sequence corresponding to SEQ ID NOS: 1 or 3, or thenucleotide sequences encode a polypeptide that is about 75 percent, orabout 80 percent, or about 85 percent, or about 90 percent, or about 95,96, 97, 98, or 99 percent identical to the polypeptide sequence as setforth in SEQ ID NOS: 2 or 4. Related nucleic acid molecules also includefragments of the above JNK activating phosphatase nucleic acid moleculeswhich are at least about 10 contiguous nucleotides, or about 15, orabout 20, or about 25, or about 50, or about 75, or about 100, orgreater than about 100 contiguous nucleotides. Related nucleic acidmolecules also include fragments of the above JNK activating phosphatasenucleic acid molecules which encode a polypeptide of at least about 25amino acid residues, or about 50, or about 75, or about 100, or greaterthan about 100 amino acid residues. Related nucleic acid molecules alsoinclude a nucleotide sequence encoding a polypeptide comprising orconsisting essentially of a substitution, modifications and/or adeletion of one to 50 amino acid residues compared to the polypeptide inSEQ ID NOS: 2 or 4. Related JNK activating phosphatase nucleic acidmolecules include those molecules that comprise nucleotide sequenceswhich hybridize under moderately or highly stringent conditions asdefined herein with any of the above nucleic acid molecules or theircomplements. In preferred embodiments, the related nucleic acidmolecules comprise sequences which hybridize under moderately or highlystringent conditions with the sequence as shown in SEQ ID NOS: 1 or 3,or with a molecule encoding a polypeptide, which polypeptide comprisesthe sequence as shown in SEQ ID NOS: 2 or 4, or with a nucleic acidfragment as defined above, or with a nucleic acid fragment encoding apolypeptide as defined above. It is also understood that related nucleicacid molecules include allelic or splice variants of any of the abovenucleic acids, and include sequences which are complementary to any ofthe above nucleotide sequences.

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule of the invention that is free from at least one contaminatingnucleic acid molecule with which it is naturally associated, andpreferably substantially free from any other contaminating mammaliannucleic acid molecules which would interfere with its use in proteinproduction or its therapeutic or diagnostic use.

The term “allelic variant” refers to one of several possible naturallyoccurring alternate forms of a gene occupying a given locus on achromosome of an organism or a population of organisms.

The term “splice variant” refers to a nucleic acid molecule, usuallyRNA, which is generated by alternative processing of intron sequences inan RNA transcript.

The term “expression vector” refers to a vector that is suitable forpropagation in a host cell and contains nucleic acid sequences thatdirect and/or control the expression of inserted heterologous nucleicacid sequences. Expression includes, but is not limited to, processessuch as transcription, translation, and RNA splicing, if introns arepresent.

The term “highly stringent conditions” refers to those conditions that(1) employ low ionic strength reagents and high temperature for washing,for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO₄ (SDS) at50° C., or (2) employ during hybridization a denaturing agent such asformamide, for example, 50% (vol/vol) formamide with 0.1% bovine serumalbumin. See Y. T. Ip and R. J. Davis, Curr. Opin. Cell Biol. 10, 205(1998); D. C. I. Goberdhan and C. Wilson, Bioassays 20, 1009 (1998).Alternatively, an example includes use of Ficoll, 0.1%polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 750 mMNaCl, and 75 mM sodium citrate at 42° C. Another example is the use of50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

The term “moderately stringent conditions” refers to conditions whichgenerally include the use of a washing solution and hybridizationconditions (e.g., temperature, ionic strength, and percentage of SDS)less stringent than described above. An example of moderately stringentconditions are conditions such as overnight incubation at 37° C. in asolution comprising 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 μl/ml denatured sheared salmon sperm DNA,followed by washing in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strengt, etc. asnecessary to accommodate factors such as probe length and the like.

In certain preferred embodiments, where oligonucleotide probes are usedto screen cDNA or genomic libraries, high stringency conditions are usedwhich depend upon the melting temperature (T_(m)) of oligonucleotideprobes to target sequences. The T_(m) may be estimated using thefollowing formula (Bolton et al., Proc. Natl. Acad. Sci. U.S.A. 48:1390(1962)):T _(m)=81.5−16.6(log[Na+])+0.41(%G+C)−(600/N)

-   -   wherein [Na+] is the sodium ion concentration in the        hybridization (or washing) solution;    -   % G+C is guanine and cytosine content in the oligonucleotide        probe; and    -   N is the probe length in nucleotides.

An example of a high stringency solution is 6×SSC and 0.05% sodiumpyrophosphate at a temperature of 35-63° C., depending on the length ofthe oligonucleotide probe. For example, according to certainembodiments, 14 base pair probes are washed at 35-40° C., 17 base pairprobes are washed at 45-50° C., 20 base pair probes are washed at 52-57°C., and 23 base pair probes are washed at 57-63° C. The temperature canbe increased 2-3° C. where the background non-specific binding appearshigh. A second high stringency solution utilizes tetramethylammoniumchloride (TMAC) for washing oligonucleotide probes. One stringentwashing solution is 3 M TMAC, 50 mM Tris-HCl, pH 8.0, and 0.2% SDS. Thewashing temperature using this solution is a function of the length ofthe probe. For example, 14 base pair probes are washed at 35-40° C., 17base pair probes are washed at about 45-50° C., 20 base pair probes arewashed at 52-57° C., and 23 base pair probes are washed at 57-63° C.

The term “JNK activating phosphatase polypeptides” refers to apolypeptide comprising the amino acid sequence of SEQ ID NOS: 1 OR 2,and related polypeptides described herein. Related polypeptides include:allelic variants; splice variants; fragments; derivatives; substitution,deletion, and insertion variants; fusion polypeptides; and orthologs.JNK activating phosphatase polypeptides may be mature polypeptides, asdefined herein, and may or may not have an amino terminal methionineresidue, depending on the method by which they are prepared.

The term “JNK activating phosphatase polypeptide fragment” refers to apeptide or polypeptide that comprises less than the full length aminoacid sequence of an JNK activating phosphatase polypeptide as set forthin SEQ ID NOS: 2 OR 4. Such a fragment may arise, for example, from atruncation at the amino terminus, a truncation at the carboxyl terminus,and/or an internal deletion of a residue(s) from the amino acidsequence. JNK activating phosphatase fragments may result fromalternative RNA splicing or from in vivo protease activity.

The term “JNK activating phosphatase polypeptide variants” refers to JNKactivating phosphatase polypeptides comprising amino acid sequenceswhich contain one or more amino acid sequence substitutions, deletions,and/or additions as compared to the JNK activating phosphatasepolypeptide amino acid sequence set forth in SEQ ID NOS: 2 or 4.Variants may be naturally occurring or artificially constructed. SuchJNK activating phosphatase polypeptide variants may be prepared from thecorresponding nucleic acid molecules encoding said variants, which havea DNA sequence that varies accordingly from the DNA sequences for wildtype JNK activating phosphatase polypeptides as set forth in SEQ ID NOS:2 or 4.

The term “JNK activating phosphatase fusion polypeptide” refers to afusion of an JNK activating phosphatase polypeptide, fragment, variant,or derivative thereof, with a heterologous peptide or polypeptide.

The term “JNK activating phosphatase polypeptide derivatives” refers toJNK activating phosphatase polypeptides, variants, or fragments thereof,that have been chemically modified, as for example, by covalentattachment of one or more polymers, including, but limited to, watersoluble polymers, N-linked or O-linked carbohydrates, sugars,phosphates, and/or other such molecules. The derivatives are modified ina manner that is different from naturally occurring JNK activatingphosphatase polypeptide, either in the type or location of the moleculesattached to the polypeptide. Derivatives further include the deletion ofone or more chemical groups naturally attached to the JNK activatingphosphatase polypeptide.

The terms “biologically active JNK activating phosphatase polypeptides,”“biologically active JNK activating phosphatase polypeptide fragments,”“biologically active JNK activating phosphatase polypeptide variants,”and “biologically active JNK activating phosphatase polypeptidederivatives” refer to JNK activating phosphatase polypeptides having atleast one activity characteristic of an JNK activating phosphatasepolypeptide, such as regulating JNK activation or mediating JNK-mediatedsignal transduction. In general, JNK activating phosphatasepolypeptides, and variants, fragments and derivatives thereof, will haveat least one activity characteristic of an JNK activating phosphatasepolypeptide such as regulating JNK activation or mediating JNK-mediatedsignal transduction. In addition, a JNK activating phosphatasepolypeptide may be active as an immunogen (i.e., the polypeptidecontains at least one epitope to which antibodies may be raised).

“Naturally occurring” when used in connection with biological materialssuch as nucleic acid molecules, polypeptides, host cells, and the like,refers to that which are found in nature and not manipulated by a humanbeing.

The term “isolated polypeptide” refers to a polypeptide of the inventionthat is free from at least one contaminating polypeptide that is foundin its natural environment, and preferably substantially free from anyother contaminating mammalian polypeptides which would interfere withits therapeutic or diagnostic use.

The term “ortholog” refers to a polypeptide that corresponds to apolypeptide identified from a different species. For example, mouse andhuman JNK activating phosphatase polypeptides are considered orthologs.

The term “mature JNK activating phosphatase polypeptide” refers to apolypeptide lacking a leader sequence and may also include othermodifications of a polypeptide such as proteolytic processing of theamino terminus (with or without a leader sequence) and/or the carboxylterminus, cleavage of a smaller polypeptide from a larger precursor,N-linked and/or O-linked glycosylation, and the like.

The terms “effective amount” and “therapeutically effective amount”refer to the amount of a JNK activating phosphatase polypeptide that isuseful or necessary to support an observable level of one or morebiological activities of the JNK activating phosphatase polypeptides asset forth above.

Relatedness of Nucleic Acid Molecules and/or Polypeptides

The term “identity,” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessbetween polypeptide or nucleic acid molecule sequences, as the case maybe, as determined by the match between strings of nucleotide or aminoacid sequences. “Identity” measures the percent of identical matchesbetween two or more sequences with gap alignments addressed by aparticular mathematical model of computer programs (i.e., “algorithms”).

The term “similarity” is a related concept, but in contrast to“identity,” refers to a measure of similarity which includes bothidentical matches and conservative substitution matches. Sinceconservative substitutions apply to polypeptides and not nucleic acidmolecules, similarity only deals with polypeptide sequence comparisons.If two polypeptide sequences have, for example, {fraction (10/20)}identical amino acids, and the remainder are all non-conservative issubstitutions, then the percent identity and similarity would both be50%. If in the same example, there are 5 more positions where there areconservative substitutions, then the percent identity remains 50%, butthe percent similarity would be 75% ({fraction (15/20)}). Therefore, incases where there are conservative substitutions, the degree ofsimilarity between two polypeptide sequences will be higher than thepercent identity between those two sequences.

The term “conservative amino acid substitution” refers to a substitutionof a native amino acid residue with a nonnative residue such that thereis little or no effect on the polarity or charge of the amino acidresidue at that position. For example, a conservative substitutionresults from the replacement of a non-polar residue in a polypeptidewith any other non-polar residue. Furthermore, any native residue in thepolypeptide may also be substituted with alanine, as has been previouslydescribed for “alanine scanning mutagenesis.” General rules forconservative amino acid substitutions are set forth in Table I. TABLE IConservative Amino Acid Substitutions Original Residues ExemplarySubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln AsnAsn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu,Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met,Ala, Phe Lys Arg, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile,Ala, Leu Tyr Pro Ala Ala Ser Thr Thr Thr Ser Ser Trp Tyr, Phe Tyr TyrTrp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, NorleucineConservative amino acid substitutions also encompass non-naturallyoccurring amino acid residues that are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics, and other reversed or invertedforms of amino acid moieties.

Conservative modifications to the amino acid sequence (and thecorresponding modifications to the encoding nucleotides) are expected toproduce JNK activating phosphatase polypeptide having functional andchemical characteristics similar to those of naturally occurring JNKactivating phosphatase polypeptide. In contrast, substantialmodifications in the functional and/or chemical characteristics of JNKactivating phosphatase polypeptide may be accomplished by selectingsubstitutions that differ significantly in their effect on maintaining(a) the structure of the molecular backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. Naturally occurring residues may be divided intogroups based on common side chain properties:

-   -   1) hydrophobic: norleucine, Met, Ala, Val, Leu, lie;    -   2) neutral hydrophilic: Cys, Ser, Thr;    -   3) acidic: Asp, Glu;    -   4) basic: Asn, Gln, His, Lys, Arg;    -   5) residues that influence chain orientation: Gly, Pro; and    -   6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced into regions of the human JNK activatingphosphatase molecule that are homologous with non-human JNK activatingphosphatase polypeptide, or into the non-homologous regions of themolecule.

Identity and similarity of related nucleic acid molecules andpolypeptides can be readily calculated by known methods, including butnot limited to those described in Computational Molecular Biology (A. M.Lesk, ed., Oxford University Press 1988); Biocomputing: Informatics andGenome Projects (D. W. Smith, ed., Academic Press 1993); ComputerAnalysis of Sequence Data (Part 1, A. M. Griffin and H. G. Griffin,eds., Humana Press 1994); G. von Heinle, Sequence Analysis in MolecularBiology (Academic Press 1987); Sequence Analysis Primer (M. Gribskov andJ. Devereux, eds., M. Stockton Press 1991); and Carillo et al., SIAM J.Applied Math., 48:1073 (1988).

Preferred methods to determine identity and/or similarity are designedto give the largest match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,Nuc. Acids Res. 12:387 (1984); Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Atschul et al., J.Mol. Biol. 215:403-10 (I990)). The BLAST X program is publicly availablefrom the National Center for Biotechnology Information (NCBI) and othersources (Altschul et al., BLAST Manual (NCB NLM NIH, Bethesda, Md.);Altschul et al., 1990, supra). The well-known Smith Waterman algorithmmay also be used to determine identity.

By way of example, using the computer algorithm GAP (Genetics ComputerGroup), two polypeptides for which the percent sequence identity is tobe determined are aligned for optimal matching of their respective aminoacids (the “matched span,” as determined by the algorithm). A gapopening penalty (which is calculated as 3× the average diagonal; the“average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid match by the particular comparison matrix) and agap extension penalty (which is usually {fraction (1/10)} times the gapopening penalty), as well as a comparison matrix such as PAM 250 orBLOSUM 62 are used in conjunction with the algorithm. A standardcomparison matrix (see Dayhoffet al., 5 Atlas of Protein Sequence andStructure (Supp. 3 1978) for the PAM250 comparison matrix; see Henikoffet al., Proc. Natl. Acad. Sci USA 89:10915-19 (1992) for the BLOSUM 62comparison matrix) is also used by the algorithm.

Preferred parameters for polypeptide sequence comparison include thefollowing:

-   -   Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-53 (1970)    -   Comparison matrix: BLOSUM 62 from Henikoffet al., Proc. Natl.        Acad. Sci. U.S.A. 89:10915-19 (1992)    -   Gap Penalty: 12    -   Gap Length Penalty: 4    -   Threshold of Similarity: 0        The GAP program is useful with the above parameters. The        aforementioned parameters are the default parameters for        polypeptide comparisons (along with no penalty for end gaps)        using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisoninclude the following:

-   -   Algorithm: Needleman et al., J. Mol Biol. 48:443-53 (1970)    -   Comparison matrix: matches=+10, mismatch=0    -   Gap Penalty: 50    -   Gap Length Penalty: 3        The GAP program is also useful with the above parameters. The        aforementioned parameters are the default paraneters for nucleic        acid molecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused by those of skill in the art, including those set forth in theProgram Manual, Wisconsin Package, Version 9, September, 1997. Theparticular choices to be made will depend on the specific comparison tobe made, such as DNA to DNA, protein to protein, protein to DNA; andadditionally, whether the comparison is between given pairs of sequences(in which case GAP or BestFit are generally preferred) or between onesequence and a large database of sequences (in which case FASTA orBLASTA are preferred).

Sequence analysis of an isolated mouse cDNA (mouse JNK activatingphosphatase-like protein; SEQ ID NO: 2) indicated that it encoded anovel member of the FGF family of proteins. The mouse JNK activatingphosphatase-like gene comprises a 615 bp open reading frame (SEQ ID NO:3) encoding a protein of 205 amino acids (SEQ ID NO: 4) (FIG. 2). Themouse sequence was used to identify the human JNK activatingphosphatase-like ortholog. Sequence analysis of a human JNK activatingphosphatase-like polypeptide cDNA clone indicated that the human JNKactivating phosphatase-like gene comprises a 615 bp open reading frame(SEQ ID NO: 1) encoding a protein of 205 amino acids (FIG. 1) (SEQ IDNO: 2).

FIG. 3(A) illustrates the amino acid sequence alignment of human JNKactivating phosphatase, mouse JNK activating phosphatase, and selectMAPK phosphatases. Computer analysis of the predicted mouse JNKactivating phosphatase-like polypeptide, using the PIMA 1.4 program ofthe Institute of Medical Genetics (URLDot.imgen.bcm.ttmc.edu:9331/multi-align/multi-align.html Baylor Collegeof Medicine Search Launcher) database, indicated that the protein wasmost closely related to HVHR. Using the GAP program, mouse JNKactivating phosphatase-like polypeptide was found to be 32% identical toHVHR The mouse JNK activating phosphatase-like polypeptide is 89%identical to the human JNK activating phosphatase-like protein.

Nucleic Acid Molecules

Recombinant DNA methods used herein are generally those set forth inSambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1989) and/or Current Protocols in MolecularBiology (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons1994).

The invention provides for nucleic acid molecules as described hereinand methods for obtaining the molecules. A gene or cDNA encoding an JNKactivating phosphatase polypeptide or fragment thereof may be obtainedby hybridization screening of a genomic or cDNA library, or by PCRamplification. Probes or primers useful for screening a library byhybridization can be generated based on sequence information for otherknown genes or gene fragments from the same or a related family ofgenes, such as, for example, conserved motifs. In addition, where a geneencoding JNK activating phosphatase polypeptide has been identified fromone species, all or a portion of that gene may be used as a probe toidentify corresponding genes from other species (orthologs) or relatedgenes from the same species (homologs). The probes or primers may beused to screen cDNA libraries from various tissue sources believed toexpress the JNK activating phosphatase gene. In addition, part or all ofa nucleic acid molecule having the sequence as set forth in SEQ ID NOS:1 or 2 may be used to screen a genomic library to identify and isolate agene encoding JNK activating phosphatase polypeptide. Typically,conditions of moderate or high stringency will be employed for screeningto minimize the number of false positives obtained from the screen.

Nucleic acid molecules encoding JNK activating phosphatase polypeptidesmay also be identified by expression cloning which employs detection ofpositive clones based upon a property of the expressed protein.Typically, nucleic acid libraries are screened by binding an antibody orother binding partner (e.g., receptor or ligand) to cloned proteinswhich are expressed and displayed on the host cell surface. The antibodyor binding partner is modified with a detectable label to identify thosecells expressing the desired clone.

Another means of preparing a nucleic acid molecule encoding an JNKactivating phosphatase polypeptide or fragment thereof is chemicalsynthesis using methods well known to the skilled artisan such as thosedescribed by Engels et al., Angew. Chem. Intl. Ed. 28:716-34 (1989).These methods include, inter alia, the phosphotriester, phosphoramidite,and H-phosphonate methods for nucleic acid synthesis. A preferred methodfor such chemical synthesis is polymer-supported synthesis usingstandard phosphoramidite chemistry. Typically, the DNA encoding the JNKactivating phosphatase polypeptide will be several hundred nucleotidesin length. Nucleic acids larger than about 100 nucleotides can besynthesized as several fragments using these methods. The fragments canthen be ligated together to form the full-length JNK activatingphosphatase polypeptide. Usually, the DNA fragment encoding the aminoterminus of the polypeptide will have an ATG, which encodes a methionineresidue. This methionine may or may not be present on the mature form ofthe JNK activating phosphatase polypeptide, depending on whether thepolypeptide produced in the host cell is designed to be secreted fromthat cell.

In some cases, it may be desirable to prepare nucleic acid moleculesencoding JNK activating phosphatase polypeptide variants. Nucleic acidmolecules encoding variants may be produced using site directedmutagenesis, PCR amplification, or other appropriate methods, where theprimer(s) have the desired point mutations (see Sanmbrook et al., supra,and Ausubel et al., supra, for descriptions of mutagenesis techniques).Chemical synthesis using methods described by Engels et al., supra, mayalso be used to prepare such variants. Other methods known to theskilled artisan may be used as well.

In certain embodiments, nucleic acid variants contain codons which havebeen altered for optimal expression of an JNK activating phosphatasepolypeptide in a given host cell. Particular codon alterations willdepend upon the JNK activating phosphatase polypeptide and host cellselected for expression. Such “codon optimization” can be carried out bya variety of methods, for example, by selecting codons which arepreferred for use in highly expressed genes in a given host cell.Computer algorithms which incorporate codon frequency tables such as“Ecohigh._Cod” for codon preference of highly expressed bacterial genesmay be used and are provided by the University of Wisconsin PackageVersion 9.0, Genetics Computer Group, Madison, Wis. Other useful codonfrequency tables include “Celegans_high.cod,” “Celegans_low.cod,”“Drosophila_high.cod,” “Human_high.cod,” “Maize_high.cod,” and“Yeast_high.cod.”

In other embodiments, nucleic acid molecules encode JNK activatingphosphatase variants with conservative amino acid substitutions asdefined above, JNK activating phosphatase variants comprising anaddition and/or a deletion of one or more N-linked or O-linkedglycosylation sites, JNK activating phosphatase variants havingdeletions and/or substitutions of one or more cysteine residues, or JNKactivating phosphatase polypeptide fragments as described above. Inaddition, nucleic acid molecules may encode any combination of JNKactivating phosphatase variants, fragments, and fusion polypeptidesdescribed herein.

Vectors and Host Cells

A nucleic acid molecule encoding an JNK activating phosphatasepolypeptide is inserted into an appropriate expression vector usingstandard ligation techniques. The vector is typically selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). A nucleic acid moleculeencoding an JNK activating phosphatase polypeptide may beamplified/expressed in prokaryotic, yeast, insect (baculovirus systems)and/or eukaryotic host cells. Selection of the host cell will depend inpart on whether an JNK activating phosphatase polypeptide is to bepost-translationally modified (e.g., glycosylated and/orphosphorylated). If so, yeast, insect, or mammalian host cells arepreferable. For a review of expression vectors, see 185 Meth. Enz. (D.V. Goeddel, ed., Academic Press 1990).

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotides: a promoter, one or moreenhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a leader sequence for secretion, a ribosomebinding site, a polyadenylation sequence, a polylinker region forinserting the nucleic acid encoding the polypeptide to be expressed, anda selectable marker element. Each of these sequences is discussed below.

Optionally, the vector may contain a “tag” sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the JNKactivating phosphatase polypeptide coding sequence; the oligonucleotidemolecule encodes polyHis (such as hexaHis), or other “tag” such as FLAG,HA (hemaglutinin Influenza virus) or myc for which commerciallyavailable antibodies exist. This tag is typically fused to thepolypeptide upon expression of the polypeptide, and can serve as a meansfor affinity purification of the JNK activating phosphatase polypeptidefrom the host cell. Affinity purification can be accomplished, forexample, by column chromatography using antibodies against the tag as anaffinity matrix. Optionally, the tag can subsequently be removed fromthe purified JNK activating phosphatase polypeptide by various meanssuch as using certain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), or synthetic, or nativesequences which normally function to regulate JNK activating phosphataseexpression. As such, the source of flanking sequences may be anyprokaryotic or eukaryotic organism, any vertebrate or invertebrateorganism, or any plant, provided that the flanking sequences isfunctional in, and can be activated by, the host cell machinery.

The flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art Typically,flanking sequences useful herein other than the JNK activatingphosphatase gene flanking sequences will have been previously identifiedby mapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of one ormore flanking sequence may be known. Here, the flanking sequence may besynthesized using the methods described above for nucleic acid synthesisor cloning.

Where all or only a portion of the flanking sequence is known, it may beobtained using PCR and/or by screening a genomic library with suitableoligonucleotide and/or flanking sequence fragments from the same oranother species.

Where the flanking sequence is not known, a fragment of DNA containing aflanking sequence may be isolated from a larger piece of DNA that maycontain, for example, a coding sequence or even another gene or genes.Isolation may be accomplished by restriction endonuclease digestion toproduce the proper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography, or other methods known tothe skilled artisan. Selection of suitable enzymes to accomplish thispurpose will be readily apparent to one of ordinary skill in the art.

An origin of replication is typically a part of prokaryotic expressionvectors purchased commercially, and aids in the amplification of thevector in a host cell. Amplification of the vector to a certain copynumber can, in some cases, be important for optimal expression of theJNK activating phosphatase polypeptide. If the vector of choice does notcontain an origin of replication site, one may be chemically synthesizedbased on a known sequence, and ligated into the vector.

The origin of replication from the plasmid pBR322 (Product No. 303-3s,New England Biolabs, Beverly, Mass.) is suitable for most Gram-negativebacteria and various origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV) or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it contains theearly promoter).

A transcription termination sequence is typically located 3′ of the endof a polypeptide coding regions and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aGE rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described above.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, for example, ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure thatonly the transformants are uniquely adapted to survive by virtue of themarker present in the vector. Selection pressure is imposed by culturingthe transformed cells under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodes JNKactivating phosphatase polypeptide. As a result, increased quantities ofJNK activating phosphatase polypeptide are synthesized from theamplified DNA.

A ribosome binding site is usually necessary for translation initiationof mma and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). the element is typically located 3′ tothe promoter and 5′ to the coding sequence of the JNK activatingphosphatase polypeptide to be expressed. the Shine-Dalgarno sequence isvaried but is typically a polypurine (i.e., having a high a-g content).many Shine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector.

In many cases, transcription of a nucleic acid molecule is increased bythe presence of one or more introns in the vector, this is particularlytrue where a polypeptide is produced in eukaryotic host cells,especially mammalian host cells. The introns used may be naturallyoccurring within the JNK activating phosphatase gene especially wherethe gene used is a full-length genomic sequence or a fragment thereof.Where the intron is not naturally occurring within the gene (as for mostcDNAs), the intron may be obtained from another source. The position ofthe intron with respect to flanking sequences and the JNK activatingphosphatase gene is generally important, as the intron must betranscribed to be effective. Thus, when an JNK activating phosphatasecDNA molecule is being expressed, the preferred position for the intronis 3′ to the transcription start site and 5′ to the poly-A transcriptiontermination sequence. Preferably, the intron or introns will be locatedon one side or the other (i.e., 5′ or 3′) of the cDNA such that it doesnot interrupt the coding sequence. Any intron from any source, includingany viral, prokaryotic and eukaryotic (plant or animal) organisms, maybe used to practice this invention, provided that it is compatible withthe host cell into which it is inserted. Also included herein aresynthetic introns. Optionally, more than one intron may be used in thevector.

The expression and cloning vectors of the present invention willtypically contain a promoter that is recognized by the host organism andoperably linked to the molecule encoding the JNK activating phosphataseprotein. Promoters are untranslated sequences located upstream (i.e.,5′) to the start codon of a structural gene (generally within about 100to 1000 bp) that control the transcription and translation of thestructural gene. Promoters are conventionally grouped into one of twoclasses: inducible promoters and constitutive promoters. Induciblepromoters initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, such asthe presence or absence of a nutrient or a change in temperature. Alarge number of promoters, recognized by a variety of potential hostcells, are well known. These promoters are operably linked to the DNAencoding JNK activating phosphatase polypeptide by removing the promoterfrom the source DNA by restriction enzyme digestion and inserting thedesired promoter sequence into the vector. The native JNK activatingphosphatase promoter sequence may be used to direct amplification and/orexpression of JNK activating phosphatase DNA. A heterologous promoter ispreferred, however, if it permits greater transcription and higheryields of the expressed protein as compared to the native promoter, andif it is compatible with the host cell system that has been selected foruse.

Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems; alkaline phosphatase, atryptophan (trp) promoter system; and hybrid promoters such as the tacpromoter. Other known bacterial promoters are also suitable. Theirsequences have been published, thereby enabling one skilled in the artto ligate them to the desired DNA sequence, using linkers or adapters asneeded to supply any required restriction sites.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude those obtained from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40). Othersuitable mammalian promoters include heterologous mammalian promoters,for example, heat-shock promoters and the actin promoter.

Additional promoters which may be of interest in controlling JNKactivating phosphatase gene expression include, but are not limited to:the SV40 early promoter region (Bernoist and Chambon, Nature 290:304-10(1981)); the CMV promoter, the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 22:787-97(1980)); the herpes thymidine kinase promoter (Wagner et al., Proc.Natl. Acad. Sci. U.S.A. 78:1444-45 (1981)); the regulatory sequences ofthe metallothionine gene (Brinster et al., Nature 296:39-42 (1982));prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Kamaroffet al., Proc. Natl. Acad. Sci. U.S.A., 75:3727-31(1978)); or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci.U.S.A., 80:21-25 (1983)). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion which is active in pancreatic acinar cells (Swift et al., Cell38:63946 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol.50:399-409 (1986); MacDonald, Hepatology 7:425-515 (1987)); the insulingene control region which is active in pancreatic beta cells (Hanahan,Nature 315:115-22 (1985)); the immunoglobulin gene control region whichis active in lymphoid cells (Grosschedl et al., Cell 38:647-58 (1984);Adames et al., Nature 318:533-38 (1985); Alexander et al., Mol. Cell.Biol., 7:1436-44 (1987)); the mouse mammary tumor virus control regionwhich is active in testicular, breast, lymphoid and mast cells (Leder etal., Cell 45:485-95 (1986)); the albumin gene control region which isactive in liver (Pinkert et al., Genes and Devel. 1:268-76 (1987)); thealpha-feto-protein gene control region which is active in liver(Krumlaufet al., Mol. Cell. Biol., 5:1639-48 (1985); Hammer etal.,Science 235:53-58 (1987)); the alpha 1-antitrypsin gene control regionwhich is active in the liver (Kelsey et al., Genes and Devel. 1: 161-71,1987)); the beta-globin gene control region which is active in myeloidcells (Mogram et al., Nature 315:338-40 (1985); Kollias et al., Cell46:89-94 (1986)); the myelin basic protein gene control region which isactive in oligodendrocyte cells in the brain (Readhead et al., Cell48:703-12 (1987)); the myosin light chain-2 gene control region which isactive in skeletal muscle (Sani, Nature 314:283-86 (1985)); and thegonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., Science 234:1372-78 (1986)).

An enhancer sequence may be inserted into the vector to increase thetranscription of a DNA encoding an JNK activating phosphatase protein ofthe present invention by higher eukaryotes. Enhancers are cis-actingelements of DNA, usually about 10-300 bp in length, that act on thepromoter to increase its transcription. Enhancers are relativelyorientation and position independent They have been found 5′ and 3′ tothe transcription unit. Several enhancer sequences available frommammalian genes are known (e.g., globin, elastase, albumin,alpha-feto-protein and insulin). Typically, however, an enhancer from avirus will be used. The SV40 enhancer, the cytomegalovirus earlypromoter enhancer, the polyoma enhancer, and adenovirus enhancers areexemplary enhancing elements for the activation of eukaryotic promoters.While an enhancer may be spliced into the vector at a position 5′ or 3′to JNK activating phosphatase DNA, it is typically located at a site 5′from the promoter.

Expression vectors of the invention may be constructed from startingvectors such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences set forth above are not already present in thevector to be used, they may be individually obtained and ligated intothe vector. Methods used for obtaining each of the flanking sequencesare well known to one skilled in the art.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA31 (Invitrogen, SanDiego, Calif.), pBSII (Stratagene, La Jolla, Calif.), pET15 (Novagen,Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2(Clontech, Palo Alto, Calif.), pETL (BlueBacII, Invitrogen), pDSR-alpha(PCT Publication No. WO90/14363) and pFastBacDual (Gibco-BRL, GrandIsland, N.Y.).

Additional possible vectors include, but are not limited to, cosmids,plasmids, or modified viruses, but the vector system must be compatiblewith the selected host cell. Such vectors include, but are not limitedto plasmids such as Bluescript® plasmid derivatives (a high copy numberColE1-based phagemid, Stratagene Cloning Systems, La Jolla Calif.), PCRcloning plasmids designed for cloning Taq-amplified PCR products (e.g.,TOPO™ TA Cloning® Kit, PCR2.1® plasmid derivatives, Invitrogen,Carlsbad, Calif.), and mammalian, yeast or virus vectors such as abaculovirus expression system (pBacPAK plasmid derivatives, Clontech,Palo Alto, Calif.). The recombinant molecules can be introduced intohost cells via transformation, transfection, infection, electroporation,or other known techniques.

After the vector has been constructed and a nucleic acid moleculeencoding an JNK activating phosphatase polypeptide has been insertedinto the proper site of the vector, the completed vector may be insertedinto a suitable host cell for amplification and/or polypeptideexpression.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast cell, an insect cell, or a vertebrate cell).The host cell, when cultured under appropriate conditions, synthesizesan JNK activating phosphatase polypeptide which can subsequently becollected from the culture medium (if the host cell secretes it into themedium) or directly from the host cell producing it (if it is notsecreted). Selection of an appropriate host cell will depend uponvarious factors, such as desired expression levels, polypeptidemodifications that are desirable or necessary for activity, such asglycosylation or phosphorylation, and ease of folding into abiologically active molecule.

A number of suitable host cells are known in the art and many areavailable from the American Type Culture Collection (ATCC), Manassas,Va. Examples include mammalian cells, such as Chinese hamster ovarycells (CHO) (ATCC No. CCL61) CHO DHFR- cells (Urlaub et al., Proc. Natl.Acad. Sci. U.S.A. 97:4216-20 (1980)), human embryonic kidney.(HEK) 293or 293T cells (ATCC No. CRL1573), or 3T3 cells (ATCC No. CCL92). Theselection of suitable mammalian host cells and methods fortransformation, culture, amplification, screening, product productionand purification are known in the art. Other suitable mammalian celllines, are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines(ATCC No. CRL1651), and the CV-1 cell line (ATCC No. CCL70). Furtherexemplary mammalian host cells include primate cell lines and rodentcell lines, including transformed cell lines. Normal diploid cells, cellstrains derived from in vitro culture of primary tissue, as well asprimary explants, are also suitable. Candidate cells may begenotypically deficient in the selection gene, or may contain adominantly acting selection gene. Other suitable mammalian cell linesinclude but are not limited to, mouse neuroblastoma N2A cells, HeLa,mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHKor HaK hamster cell lines (ATCC). Each of these cell lines is known byand available to those skilled in the art of protein expression.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, ATCC No. 33694; DH5; DH10; and MC1061, ATCC No. 53338) are wellknown as host cells in the field of biotechnology. Various strains of B.subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp., andthe like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention. Preferred yeast cells include, for example,Saccharomyces cerivisae.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described, forexample, in Kitts et al., Biotechniques, 14:8 10-17 (1993); Lucklow,Curr. Opin. Biotechnol. 4:564-72 (1993); and Lucklow et al., J. Virol.,67:4566-79 (1993). Preferred insect cells are Sf-9 and Hi5 (Invitrogen).

Transformation or transfection of an expression vector for an JNKactivating phosphatase polypeptide into a selected host cell may beaccomplished by well known methods including methods such as calciumchloride, electroporation, microinjection, lipofection or theDEAE-dextran method. The method selected will in part be a function ofthe type of host cell to be used. These methods and other suitablemethods are well known to the skilled artisan, and are set forth, forexample, in Sambrook et al., supra.

Polypeptide Production

Host cells comprising an JNK activating phosphatase polypeptideexpression vector (i.e., transformed or transfected) may be culturedusing standard media well known to the skilled artisan. The media willusually contain all nutrients necessary for the growth and survival ofthe cells. Suitable media for culturing E. coli cells are for example,Luria Broth (LB) and/or Terrific Broth (TB). Suitable media forculturing eukaryotic cells are RPMI 1640, MEM, DMEM, all of which may besupplemented with serum and/or growth factors as required by theparticular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

Typically, an antibiotic or other compound useful for selective growthof transfected or transformed cells is added as a supplement to themedia. The compound to be used will be dictated by the selectable markerelement present on the plasmid with which the host cell was transformed.For example, where the selectable marker element is kanamycinresistance, the compound added to the culture medium will be kanauycin.Other compounds for selective growth include ampicillin, tetracycline,and neomycin.

The amount of an JNK activating phosphatase polypeptide produced by ahost cell can be evaluated using standard methods known in the art. Suchmethods include, without limitation, Western blot analysis,SDS-polyacrylamide gel electrophoresis, non-denaturing gelelectrophoresis, HPLC separation, immunoprecipitation, and/or activityassays such as DNA binding gel shift assays.

If an JNK activating phosphatase polypeptide has been designed to besecreted from the host cells, the majority of polypeptide may be foundin the cell culture medium. If however, the JNK activating phosphatasepolypeptide is not secreted from the host cells, it will be present inthe cytoplasm and/or the nucleus (for eukaryotic host cells) or in thecytosol (for gram-negative bacteria host cells).

For an JNK activating phosphatase polypeptide situated in the host cellcytoplasm and/or nucleus, the host cells are typically first disruptedmechanically or with detergent to release the intra-cellular contentsinto a buffered solution. JNK activating phosphatase polypeptide canthen be isolated from this solution.

Purification of an JNK activating phosphatase polypeptide from solutioncan be accomplished using a varxiety of techniques. If the polypeptidehas been synthesized such that it contains a tag such as Hexahistidine(JNK activating phosphatase polypeptide/hexaHis) or other small peptidesuch as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen)at either its carboxyl or amino terminus, it may essentially be purifiedin a one-step process by passing the solution through an affinity columnwhere the column matrix has a high affinity for the tag or for thepolypeptide directly (i.e., a monoclonal antibody specificallyrecognizing JNK activating phosphatase polypeptide). For example,polyhistidine binds with great affinity and specificity to nickel andthus an affinity column of nickel (such as the Qiagen® nickel columns)can be used for purification of JNK activating phosphatasepolypeptide/polyHis. See, e.g., Current Protocols in Molecular Biology§10.11.8 (Ausubel et al., eds., John Wiley & Sons 1993).

Where an JNK activating phosphatase polypeptide is prepared without atag attached, and no antibodies are available, other well-knownprocedures for purification can be used. Such procedures include,without limitation, ion exchange chromatography, molecular sievechromatography, HPLC, native gel electrophoresis in combination with gelelution, and preparative isoelectric focusing (“Isoprime”machine/technique, Hoefer Scientific). In some cases, two or more ofthese techniques may be combined to achieve increased purity.

If an JNK activating phosphatase polypeptide is producedintracellularly, the intracellular material (including inclusion bodiesfor gram-negative bacteria) can be extracted from the host cell usingany standard technique known to the skilled artisan. For example, thehost cells can be lysed to release the contents of theperiplasm/cytoplasm by French press, homogenization, and/or sonicationfollowed by centrifugation.

If an JNK activating phosphatase polypeptide has formed inclusion bodiesin the cytosol, the inclusion bodies can often bind to the inner and/orouter cellular membranes and thus will be found primarily in the pelletmaterial after centrifugation. The pellet material can then be treatedat pH extremes or with chaotropic agent such as a detergent, guanidine,guanidine derivatives, urea, or urea derivatives in the presence of areducing agent such as dithiothreitol at alkaline pH or triscarboxyethyl phosphine at acid pH to release, break apart, andsolubilize the inclusion bodies. The JNK activating phosphatasepolypeptide in its now soluble form can then be analyzed using gelelectrophoresis, immunoprecipitation, or the like. If it is desired toisolate the JNK activating phosphatase polypeptide, isolation may beaccomplished using standard methods such as those set forth below and inMarston et al., Meth. Enz., 182:264-75 (1990).

In some cases, an JNK activating phosphatase polypeptide may not bebiologically active upon isolation. Various methods for “refolding” orconverting the polypeptide to its tertiary structure and generatingdisulfide linkages can be used to restore biological activity. Suchmethods include exposing the solubilized polypeptide to a pH usuallyabove 7 and in the presence of a particular concentration of achaotrope. The selection of chaotrope is very similar to the choicesused for inclusion body solubilization, but usually the chaotrope isused at a lower concentration and is not necessarily the same aschaotropes used for the so lubilization. In most cases therefoldingtoxidation solution will also contain a reducing agent or thereducing agent plus its oxidized form in a specific ratio to generate aparticular redox potential allowing for disulfide shuffling to occur inthe formation of the protein's cysteine bridges. Some of the commonlyused redox couples include cysteine/cystamine, glutathione(GSH)/dithiobis GSH, cupric chloride, dithiothbeitol(DTT)/dithiane DOT,and 2-mercaptoethanol(bME)/dithio-b(ME). In many instances, a cosolventmay be used or may be needed to increase the efficiency of the refoldingand the more common reagents used for this purpose include glycerol,polyethylene glycol of various molecular weights, arginine and the like.

If inclusion bodies are not formed to a significant degree uponexpression of an JNK activating phosphatase polypeptide, the polypeptidewill be found primarily in the supernatant after centrifugation of thecell homogenate and may be further isolated from the supernatant usingmethods such as those set forth below.

In situations where it is preferable to partially or completely purifyan JNK activating phosphatase polypeptide such that it is partially orsubstantially free of contaminants, standard methods known to the oneskilled in the art may be used. Such methods include, withoutlimitation, separation by electrophoresis followed by electroelution,various types of chromatography (affinity, immunoaffinity, molecularsieve, and/or ion exchange), and/or high pressure liquid chromatography.In some cases, it may be preferable to use more than one of thesemethods for complete purification.

JNK activating phosphatase polypeptides, fragments, and/or derivativesthereof may also be prepared by chemical synthesis methods (such assolid phase peptide synthesis) using techniques known in the art such asthose set forth by Merrifield et al., J. Am. Chem. Soc. 85:2149 (1963);Houghten et al., Proc Natl Acad. Sci. USA 82:5132 (1985); and Stewartand Young, Solid Phase Peptide Synthesis (Pierce Chemical Co. 1984).Such polypeptides may be synthesized with or without a methionine on theamino terminus. Chemically synthesized JNK activating phosphatasepolypeptides or fragments may be oxidized using methods set forth inthese references to form disulfide bridges. JNK activating phosphatasepolypeptides, fragments or derivatives are expected to have comparablebiological activity to the corresponding JNK activating phosphatasepolypeptides, fragments or derivatives produced recombinantly orpurified from natural sources, and thus may be used interchangeably withrecombinant or natural JNK activating phosphatase polypeptide.

Another means of obtaining JNK activating phosphatase polypeptide is viapurification from biological samples such as source tissues and/orfluids in which the JNK activating phosphatase polypeptide is naturallyfound. Such purification can be conducted using methods for proteinpurification as described above. The presence of the JNK activatingphosphatase polypeptide during purification may be monitored using, forexample, an antibody prepared against recombinantly produced JNKactivating phosphatase polypeptide or peptide fragments thereof.

Polypeptides

Polypeptides of the invention include isolated JNK activatingphosphatase polypeptides and polypeptides related thereto includingfragments, variants, fusion polypeptides, and derivatives as definedhereinabove.

JNK activating phosphatase polypeptide fragments of the invention mayresult from truncations at the amino terminus (with or without a leadersequence), truncations at the carboxyl terminus, and/or deletionsinternal to the polypeptide. In preferred embodiments, truncationsand/or deletions comprise about 10 amino acids, or about 20 amino acid,or about 50 amino acids, or about 75 amino acids, or about 100 aminoacids, or more than about 100 amino acids. The polypeptide fragments soproduced will comprise about 25 contiguous amino acids, or about 50amino acids, or about 75 aminio acids, or about 100 amino acids, orabout 150 amino acids, or about 200 amino acids. Such JNK activatingphosphatase polypeptide fragments may optionally comprise an aminoterminal methionine residue.

JNK activating phosphatase polypeptide variants of the invention includeone or more amino acid substitutions, additions and/or deletions ascompared to SEQ ID NOS: 2 or 4. In preferred embodiments, the variantshave from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, orfrom 1 to 20, or from 1 to 25, or from 1 to 50, or from 1 to 75, or from1 to 100, or more than 100 amino acid substitutions, insertions,additions and/or deletions, wherein the substitutions may beconservative, as defined above, or non-conservative or any combinationthereof. The variants may have additions of amino acid residues eitherat the carboxyl terminus or at the amino terminus (with or without aleader sequence).

Preferred JNK activating phosphatase variants include cysteine variants,wherein one or more cysteine residues are deleted or substituted withanother amino acid (e.g., serine). Cysteine variants are useful when JNKactivating phosphatase polypeptide must be refolded into a biologicallyactive conformation such as after isolation of insoluble inclusivebodies. Cysteine variants generally have fewer cysteine residues thanthe native protein, and typically have an even number to minimizeinteractions resulting from unpaired cysteines.

One skilled in the art will be able to determine suitable variants ofthe native JNK activating phosphatase polypeptide using well-knowntechniques. For example, one may be able to predict suitable areas ofthe molecule that may be changed without destroying biological activity.Also, one skilled in the art will realize that even areas that may beimportant for biological activity or for structure may be subject toconservative amino acid substitutions without destroying the biologicalactivity or without adversely affecting the polypeptide structure.

For predicting suitable areas of the molecule that may be changedwithout destroying activity, one skilled in the art may target areas notbelieved to be important for activity. For example, when similarpolypeptides with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of JNK activating phosphatase polypeptide to such similarpolypeptides. After making such a comparison, one skilled in the artwould be able to determine residues and portions of the molecules thatare conserved among similar polypeptides. One skilled in the art wouldknow that changes in areas of the JNK activating phosphatase moleculethat are not conserved would be less likely to adversely affectbiological activity and/or structure. One skilled in the art would alsoknow that, even in relatively conserved regions, one could have likelysubstituted chemically similar amino acids for the naturally occurringresidues while retaining activity (conservative amino acid residuesubstitutions).

Also, one skilled in the art may review structure-function studiesidentifying residues in similar polypeptides that are important foractivity or structure. In view of such a comparison, one skilled in theart can predict the importance of amino acid residues in JNK activatingphosphatase polypeptide that correspond to amino acid residues that areimportant for activity or structure in similar polypeptides. One skilledin the art may opt for chemically similar amino acid substitutions forsuch predicted important amino acid residues of JNK activatingphosphatase polypeptide.

If available, one skilled in the art can also analyze thethree-dimensional structure and amino acid sequence in relation to thatstructure in similar polypeptides. In view of that information, oneskilled in the art may be able to predict the alignment of amino acidresidues of JNK activating phosphatase polypeptide with respect to itsthree dimensional structure. One skilled in the art may choose not tomake radical changes to amino acid residues predicted to be on thesurface of the protein, since such residues may be involved in importantinteractions with other molecules.

Moreover, one skilled in the art could generate test variants containinga single amino acid substitution at each amino acid residue. Thevariants could be screened using activity assays disclosed in thisapplication. Such variants could be used to gather information aboutsuitable variants. For example, if one discovered that a change to aparticular amino acid residue resulted in destroyed activity, variantswith such a change would be avoided. In other words, based oninformation gathered from such experiments, when attempting to findadditional acceptable variants, one skilled in the art would have knownthe amino acids where further substitutions should be avoided eitheralone or in combination with other mutations.

JNK activating phosphatase fusion polypeptides of the invention compriseJNK activating phosphatase polypeptides, fragments, variants, orderivatives fused to a heterologous peptide or protein. Heterologouspeptides and proteins include, but are not limited to: an epitope toallow for detection and/or isolation of an JNK activating phosphatasefusion polypeptide; a transmembrane receptor protein or a portionthereof, such as an extracellular domain, or a transmembrane andintracellular domain; a ligand or a portion thereof which binds to atransmembrane receptor protein; an enzyme or portion thereof which iscatalytically active; a protein or peptide which promotesoligomerization, such as leucine zipper domain; and a protein or peptidewhich increases stability, such as an immunoglobulin constant region. AnJNK activating phosphatase polypeptide may be fused to itself or to afragment, variant, or derivative thereof. Fusions may be made either atthe amino terminus or at the carboxyl terminus of an JNK activatingphosphatase polypeptide, and may be direct with no linker or adaptermolecule or may be through a linker or adapter molecule, such as one ormore amino acid residues up to about 20 amino acids residues, or up toabout 50 amino acid residues. A linker or adapter molecule may also bedesigned with a cleavage site for a DNA restriction endonuclease or fora protease to allow for separation of the fused moieties.

In a further embodiment of the invention, an JNK activating phosphatasepolypeptide, fragment, variant and/or derivative is fused to an Fcregion of human IgG. In one example, a human IgG hinge, CH2 and CH3region may be fused at either the N-terminus or C-terminus of the JNKactivating phosphatase polypeptides using methods known to the skilledartisan. In another example, a portion of a hinge regions and CH2 andCH3 regions may be fused. The JNK activating phosphatase Fc-fusionpolypeptide so produced may be purified by use of a Protein A affinitycolumn. In addition, peptides and proteins fused to an Fc region havebeen found to exhibit a substantially greater half-life in vivo than theunfused counterpart. Also, a fusion to an Fc region allows fordimerization/multimerization of the fusion polypeptide. The Fc regionmay be a naturally occurring Fc region, or may be altered to improvecertain qualities, such as therapeutic qualities, circulation time,reduced aggregation, etc.

JNK activating phosphatase polypeptide derivatives are included in thescope of the present invention. Such derivatives are chemically modifiedJNK activating phosphatase polypeptide compositions in which JNKactivating phosphatase polypeptide is linked to a polymer. The polymerselected is typically water-soluble so that the protein to which it isattached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be of any molecular weight,and may be branched or unbranched. Included within the scope of JNKactivating phosphatase polypeptide polymers is a mixture of polymers.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable.

The water soluble polymer or mixture thereof may be selected from thegroup consisting of, for example, polyethylene glycol (PEG),monomethoxy-polyethylene glycol, dextran (such as low molecular weightdextran, of, for example about 6 kD), cellulose, or other carbohydratebased polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol,propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinylalcohol. Also encompassed by the invention are bifunctional PEGcross-linking molecules that may be used to prepare covalently attachedJNK activating phosphatase polypeptide multimers

For the acylation reactions, the polymer(s) selected should have asingle reactive ester group. For reductive alkylation, the polymer(s)selected should have a single reactive aldehyde group. A reactivealdehyde is, for example, polyethylene glycol propionaldehyde, which iswater stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (seeU.S. Pat. No. 5,252,714).

Pegylation of JNK activating phosphatase polypeptides may be carried outby any of the pegylation reactions known in the art, as described forexample in the following references: Francis et al., Focus on GrowthFactors 3, 4-10 (1992); EP 0154 316; EP 0 401 384 and U.S. Pat. No.4,179,337. Pegylation may be carried out via an acylation reaction or analkylation reaction with a reactive polyethylene glycol molecule (or ananalogous reactive water-soluble polymer) as described below.

One water-soluble polymer for use herein is polyethylene glycol,abbreviated PEG. As used herein, polyethylene glycol is meant toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono-(C1-C₁₀) alkoxy- or aryloxy-polyethyleneglycol.

In general, chemical derivatization may be performed under any suitableconditions used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated JNKactivating phosphatase polypeptides will generally comprise the steps of(a) reacting the polypeptide with polyethylene glycol (such as areactive ester or aldehyde derivative of PEG) under conditions wherebyJNK activating phosphatase polypeptide becomes attached to one or morePEG groups, and (b) obtaining the reaction product(s). In general, theoptimal reaction conditions for the acylation reactions will bedetermined based on known parameters and the desired result. Forexample, the larger the ratio of PEG: protein, the greater thepercentage of poly-pegylated product.

In a preferred embodiment, the JNK activating phosphatase polypeptidederivative will have a single PEG moiety at the amino terminus. See U.S.Pat. No. 5,234,784, herein incorporated by reference.

Generally, conditions that may be alleviated or modulated byadministration of the present JNK activating phosphatase polypeptidederivative include those described herein for JNK activating phosphatasepolypeptides. However, the JNK activating phosphatase polypeptidederivative disclosed herein may have additional activities, enhanced orreduced biological activity, or other characteristics, such as increasedor decreased half-life, as compared to the non-derivatized molecules.

Antibodies

JNK activating phosphatase polypeptides, fragments, variants, andderivatives may be used to prepare antibodies using methods known in theart. Thus, antibodies and antibody fragments that bind JNK activatingphosphatase polypeptides are within the scope of the present invention.Antibodies may be polyclonal, monospecific polyclonal, monoclonal,recombinant, chimeric, humanized, fully human, single chain and/orbispecific.

Polyclonal antibodies directed toward an JNK activating phosphatasepolypeptide generally are raised in animals (e.g., rabbits or mice) bymultiple subcutaneous or intraperitoneal injections of JNK activatingphosphatase polypeptide and an adjuvant It may be useful to conjugate anJNK activating phosphatase polypeptide, or a variant, fragment orderivative thereof to a carrier protein that is immunogenic in thespecies to be immunized, such as keyhole limpet heocyanin, serum,albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also,aggregating agents such as alum are used to enhance the immune response.After immunization, the animals are bled and the serum is assayed foranti-JNK activating phosphatase antibody titer.

Monoclonal antibodies directed toward JNK activating phosphatasepolypeptide are produced using any method that provides for theproduction of antibody molecules by continuous cell lines in culture.Examples of suitable methods for preparing monoclonal antibodies includehybridoma methods of Kohler, et al., Nature 256:495-97 (1975), and thehuman B-cell hybridoma method, Kozbor, J. Immunol. 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications 51-63 (Marcel Dekker 1987).

Also provided by the invention are hybridoma cell lines that producemonoclonal antibodies reactive with JNK activating phosphatasepolypeptides. Monoclonal antibodies of the invention may be modified foruse as therapeutics. One embodiment is a “chimeric” antibody in which aportion of the heavy and/or light chain is identical with or homologousto corresponding sequence in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is identical with or homologous tocorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(see U.S. Pat. No. 4,8 16,567; Morrison, et al., Proc. Natl. Acad. Sci.U.S.A. 81: 6851-55 (1985).

In another embodiment, a monoclonal antibody of the invention is a“humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source that is non-human.Humanization can be performed following methods known in the art (Jones,et al., Nature 321: 522-25 (1986); Riechmann, et al., Nature 332:323-27(1988); Verhoeyen et al., Science 239:1534-36 (1988)), by substitutingrodent complementarily-determining regions (CDRs) for the-correspondingregions of a human antibody.

Also encompassed by the invention are fully human antibodies that bindJNK activating phosphatase polypeptides, fragments, variants, and/orderivatives. Such antibodies are produced by immunization with an JNKactivating phosphatase antigen (optionally conjugated to a carrier) oftransgenic animals (e.g., mice) that are capable of producing arepertoire of human antibodies in the absence of endogenousimmunoglobulin production. See, e.g., Jakobovits, et al., Proc. Natl.Acad. Sci. U.S.A 90: 2551-55 (1993); Jakobovits, et al., Nature362:255-58 (1993); Bruggermann et al., Year in Immuno. 7:33 (1993).Human antibodies can also be produced in phagedisplay libraries(Hoogenboom et al., J. Mol. Biol. 227:381 (1991); Marks, et al., J. Mol.Biol. 222:581 (1991)).

The anti-JNK activating phosphatase antibodies of the invention may beemployed in any known assay method, such as competitive binding assays,direct and indirect sandwich assays, and immunoprecipitation assays(Sola, Monoclonal Antibodies: A Manual of Techniques 147-58 (CRC Press1987)) for detection and quantitation of JNK activating phosphatasepolypeptides. The antibodies will bind JNK activating phosphatasepolypeptides with an affinity that is appropriate for the assay methodbeing employed.

Competitive binding assays rely on the ability of a labeled standard(e.g., an JNK activating phosphatase polypeptide, or an immunologicallyreactive portion thereof) to compete with the test sample analyte (anJNK activating phosphatase polypeptide) for binding with a limitedamount of anti JNK activating phosphatase antibody. The amount of an JNKactivating phosphatase polypeptide in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies typically are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected and/or quantitated. In a sandwich assay, the test sampleanalyte is typically bound by a first antibody which is immobilized on asolid support, and thereafter a second antibody binds to the analyte,thus forming an insoluble three part complex. See, e.g., U.S. Pat. No.4,376,110. The second antibody may itself be labeled with a detectablemoiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan ELISA assay, in which case the detectable moiety is an enzyme.

The invention also relates to a kit comprising anti-JNK activatingphosphatase antibodies and other reagents useful for detecting JNKactivating phosphatase polypeptide levels in biological samples. Suchreagents may include a secondary activity, a detectable label, blockingserum, positive and negative control samples and detection reagents.

Genetically Engineered Non-Human Animals

Additionally included within the scope of the present invention arenon-human animals such as mice, rats, or other rodents, rabbits, goats,sheep, or other farm animals, in which the gene encoding native JNKactivating phosphatase polypeptide has been disrupted (i.e., “knockedout”) such that the level of expression of JNK activating phosphatasepolypeptide is significantly decreased or completely abolished. Suchanimals may be prepared using techniques and methods such as thosedescribed in U.S. Pat. No. 5,557,032.

The present invention further includes non-human animals such as mice,rats, or other rodents, rabbits, goats, sheep, or other farm animals, inwhich a gene encoding a native form of JNK activating phosphatasepolypeptide for that animal or a heterologous JNK activating phosphatasepolypeptide gene is overexpressed by the animal, thereby creating a“transgenic” animal. Such transgenic animals may be prepared using wellknown methods such as those described in U.S. Pat. No 5,489,743 and PCTPublication No. WO94/28122.

The present invention further includes non-human animals in which thepromoter for one or more of the JNK activating phosphatase polypeptidesof the present invention is either activated or inactivated (e.g., byusing homologous recombination methods as described below) to alter thelevel of expression of one or more of the native JNK activatingphosphatase polypeptides.

Such non-human animals may be used for drug candidate screening. Theimpact of a drug candidate on the animal may be measured. For example,drug candidates may decrease or increase expression of the JNKactivating phosphatase polypeptide gene. In certain embodiments, theamount of JNK activating phosphatase polypeptide or an JNK activatingphosphatase polypeptide fragment that is produced may be measured afterexposure of the animal to the drug candidate. In certain embodiments,one may detect the actual impact of the drug candidate on the animal.For example, overexpression of a particular gene may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease expression of the geneor its ability to prevent or inhibit a pathological condition. In otherexamples, production of a particular metabolic product such as afragment of a polypeptide, may result in, or be associated with, adisease or pathological condition. In such cases, one may test a drugcandidate's ability to decrease production of such a metabolic productor its ability to prevent or inhibit a pathological condition.

Modulators of JNK Activating Phosphatase Polypeptide Activity

In some situations, it may be desirable to identify molecules that aremodulators, i.e., agonists or antagonists, of the activity of JNKactivating phosphatase polypeptide.

Natural or synthetic molecules that modulate JNK activating phosphatasepolypeptide can be identified using one or more screening assays, suchas those described below. Such molecules may be administered either inan ex vivo manner or in an in vivo manner by local or intravenousinjection or by oral delivery, implantation device, or the like.

The following definition is used herein for describing the assays:

“Test molecule(s)” refers to the molecule(s) that is/are underevaluation for the ability to modulate (i.e., increase or decrease) theactivity of an JNK activating phosphatase polypeptide. Most commonly, atest molecule will interact directly with an JNK activating phosphatasepolypeptide. However, it is also contemplated that a test molecule mayalso modulate JNK activating phosphatase polypeptide activityindirectly, such as by affecting JNK activating phosphatase geneexpression, or by binding to an JNK activating phosphatase bindingpartner (e.g., receptor or ligand). In one embodiment, test moleculewill bind to an JNK activating phosphatase polypeptide with an affinityconstant of at least about 10⁻⁶ M, preferably about 10⁻⁸ M, morepreferably about 10⁻⁹ M, and even more preferably about 10⁻¹⁰ M.

Methods for identifying compounds that interact with JNK activatingphosphatase polypeptides are encompassed by the invention. In certainembodiments, an JNK activating phosphatase polypeptide is incubated witha test molecule under conditions that permit interaction of the testmolecule with an JNK activating phosphatase polypeptide, and the extentof the interaction can be measured. The test molecule may be screened ina substantially purified form or in a crude mixture. Test molecules maybe nucleic acid molecules, proteins, peptides, carbohydrates, lipids, orsmall molecular weight organic or inorganic compounds. Once a set oftest molecules has been identified as interacting with an JNK activatingphosphatase polypeptide, the molecules may be further evaluated fortheir ability to increase or decrease JNK activating phosphatasepolypeptide activity.

Measurement of the interaction of test molecules with JNK activatingphosphatase polypeptides may be carried out in several formats,including cell-based binding assays, membrane binding assays,solution-phase assays and immunoassays. In general, test molecules areincubated with an JNK activating phosphatase polypeptide for a specifiedperiod of time and JNK activating phosphatase polypeptide activity isdetermined by one or more assays described herein for measuringbiological activity.

Interaction of test molecules with JNK activating phosphatasepolypeptides may also be assayed directly using polyclonal or monoclonalantibodies in an immunoassay Alternatively, modified forms of JNKactivating phosphatase polypeptides containing epitope tags as describedabove may be used in solution and immunoassays.

In certain embodiments, an JNK activating phosphatase polypeptideagonist or antagonist may be a protein, peptide, carbohydrate, lipid, orsmall molecular weight molecule that interacts with JNK activatingphosphatase polypeptide to regulate its activity. Potential proteinantagonists of JNK activating phosphatase polypeptide include antibodiesthat interest with active regions of the polypeptide and inhibit oreliminate at least one activity of JNK activating phosphatasepolypeptide. Molecules which regulate JNK activating phosphatasepolypeptide expression may include nucleic acids which are complementaryto nucleic acids encoding an JNK activating phosphatase polypeptide, orare complementary to nucleic acids sequences which direct or controlexpression of JNK activating phosphatase polypeptide, and which act asanti-sense regulators of expression.

In the event that JNX activating phosphatase polypeptides displaybiological activity through interaction with a binding partner (e.g., areceptor or a ligand), a variety of in vitro assays may be used tomeasure binding of an JNK activating phosphatase polypeptide to acorresponding binding partner. These assays may be used to screen testmolecules for their ability to increase or decrease the rate and/or theextent of binding of an JNK activating phosphatase polypeptide to itsbinding partner. In one assay, an JNK activating phosphatase polypeptideis immobilized by attachment to the bottom of the wells of a microtiterplate. Radiolabeled JNK activating phosphatase binding partner (forexample, iodinated JNK activating phosphatase binding partner) and thetest molecules can then be added either one at a time (in either order)or simultaneously to the wells. After incubation, the wells can bewashed and counted using a scintillation counter for radioactivity todetermine the extent of binding to JNK activating phosphatase protein byits binding partner. Typically, the molecules will be tested over arange of concentrations and a series of control wells lacking one ormore elements of the test assays can be used for accuracy in evaluationof the results. An alternative to this method involves reversing the“positions” of the proteins, i.e., immobilizing JNK activatingphosphatase binding partner to the microtiter plate wells, incubatingwith the test molecule and radiolabeled JNK activating phosphatasepolypeptide, and determining the extent of JNK activating phosphatasebinding (see, e.g., Current Protocols in Molecular Biology, chap. 18(Ausubel et al., eds., John Wiley & Sons 1995)).

As an alternative to radiolabelling, an JNK activating phosphatasepolypeptide or its binding partner may be conjugated to biotin and thepresence of biotinylated protein can then be detected using streptavidinlinked to an enzyme, such as horse radish peroxidase (UP) or alkalinephosphatase (AP), that can be detected colorometrically, or byfluorescent tagging of streptavidin. An antibody directed to an JNKactivating phosphatase polypeptide or to an JNK activating phosphatasebinding partner and that is conjugated to biotin may also be used andcan be detected after incubation with enzyme-linked streptavidin linkedto AP or HRP

An JNK activating phosphatase polypeptide and an JNK activatingphosphatase binding partner may also be immobilized by attachment toagarose beads, acrylic beads, or other types of such inert substrates.The substrate-protein complex can be placed in a solution containing thecomplementary protein and the test compound; after incubation, the beadscan be precipitated by centrifugation, and the amount of binding betweenan JNK activating phosphatase polypeptide and its binding partner can beassessed using the methods described above. Alternatively, thesubstrate-protein complex can be immobilized in a column and the testmolecule and complementary protein passed over the column. Formation ofa complex between an JNK activating phosphatase polypeptide and itsbinding partner can then be assessed using any of the techniques setforth above, i.e., radiolabeling, antibody binding, or the like.

Another in vitro assay that is useful for identifying a test moleculewhich increases or decreases formation of a complex between an JNKactivating phosphatase binding protein and an JNK activating phosphatasebinding partner is a surface plasmon resonance detector system such asthe Biacore assay system (Pharmacia, Piscataway, N.J.). The Biacoresystem may be carried out using the manufacturer's protocol. This assayessentially involves covalent binding of either JNK activatingphosphatase polypeptide or an JNK activating phosphatase binding partnerto a dextran-coated sensor chip that is located in a detector. The testcompound and the other complementary protein can then be injected intothe chamber containing the sensor chip either simultaneously orsequentially and the amount of complementary protein that binds can beassessed based on the change in molecular mass which is physicallyassociated with the dextranuoated side of the sensor chip; the change inmolecular mass can be measured by the detector system.

In some cases, it may be desirable to evaluate two or more testcompounds together for their ability to increase or decrease formationof a complex between an JNK activating phosphatase polypeptide and anJNK activating phosphatase binding partner complex. In these cases, theassays set forth above can be readily modified by adding such additionaltest compounds either simultaneous with, or subsequent to, the firsttest compound. The remaining steps in the assay are as set forth above.

In vitro assays such as those described above may be used advantageouslyto screen rapidly large numbers of compounds for effects on complexformation by JNK activating phosphatase polypeptide and JNK activatingphosphatase binding partner. The assays may be automated to screencompounds generated in phage display, synthetic peptide, and chemicalsynthesis libraries.

Compounds which increase or decrease formation of a complex between anJNK activating phosphatase polypeptide and an JNK activating phosphatasebinding partner may also be screened in cell culture using cells andcell lines expressing either JNK activating phosphatase polypeptide orJNK activating phosphatase binding partner. Cells and cell lines may beobtained from any mammal, but preferably will be from human or otherprimate, canine, or rodent sources.

Cell cultures can also be used to screen the impact of a drug candidate.For example, drug candidates may decrease or increase expression of theJNK activating phosphatase polypeptide gene. In certain embodiments, theamount of JNK activating phosphatase polypeptide or an JNK activatingphosphatase polypeptide fragment that is produced may be measured afterexposure of the cell culture to the drug candidate. In certainembodiments, one may detect the actual impact of the drug candidate onthe cell culture. For example, overexpression of a particular gene mayhave a particular impact on the cell culture. In such cases, one maytest a drug candidate's ability to increase or decrease expression ofthe gene or its ability to prevent or inhibit a particular impact on thecell culture. In other examples, production of a particular metabolicproduct such as a fragment of a polypeptide, may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease production of such ametabolic product in a cell culture.

A yeast two hybrid system (Chien et al., Proc. Natl. Acad. Sci. U.S.A.88:9578-83 (1991)) can be used to identify novel polypeptides that bindto, or interact with, JNK activating phosphatase polypeptides. As anexample, hybrid constructs comprising DNA encoding a cytoplasmic domainof an JNK activating phosphatase polypeptide fused to a yeast GAL4-DNAbinding domain may be used as a two-hybrid bait plasmid. Positive clonesemerging from the screening may be characterized further to identifyinteracting proteins.

Additional objects of the present invention relate to methods for boththe in vitro production of therapeutic proteins by means of homologousrecombination and for the production and delivery of therapeuticproteins by gene therapy.

It is further envisioned that JNK activating phosphatase protein may beproduced by homologous recombination, or with recombinant productionmethods utilizing control elements introduced into cells alreadycontaining DNA encoding JNK activating phosphatase polypeptide. Forexample, homologous recombination methods may be used to modify a cellthat contains a normally transcriptionally silent JNK activatingphosphatase gene, or under expressed gene, and thereby produce a cellthat expresses therapeutically efficacious amounts of JNK activatingphosphatase polypeptide. Homologous recombination is a techniqueoriginally developed for targeting genes to induce or correct mutationsin transcriptionally active genes (Kucherlapati, Prog. in Nucl. AcidRes. and Mol. Biol. 36:301 (1989)). The basic technique was developed asa method for introducing specific mutations into specific regions of themammalian genome (Thomas et al., Cell 44:419-28 (1986); Thomas andCapecchi, Cell 51:503-12, (1987); Doetschman et al., Proc. Natl. Acad.Sci. U.S.A. 85:8583-87 (1988)) or to correct specific mutations withindefective genes (Doetschman et al., Nature 330:576-78 (1987)). Exemplaryhomologous recombination techniques are described in U.S. Pat. No.5,272,071 (EP Patent No. 91 90 3051, EP Publication No. 505 500;PCT/US90/07642, International Publication No. WO 91/09955).

Through homologous recombination, the DNA sequence to be inserted intothe genome can be directed to a specific region of the gene of interestby attaching it to targeting DNA. The targeting DNA is a nucleotidesequence that is complementary (homologous) to a region of the genomicDNA. Small pieces of targeting DNA that are complementary to a specificregion of the genome are put in contact with the parental strand duringthe DNA replication process. It is a general property of DNA that hasbeen inserted into a cell to hybridize, and therefore, recombine withother pieces of endogenous DNA through shared homologous regions. Ifthis complementary strand is attached to an oligonucleotide thatcontains a mutation or a different sequence or an additional nucleotide,it too is incorporated into the newly synthesized strand as a result ofthe recombination. As a result of the proofreading function, it ispossible for the new sequence of DNA to serve as the template. Thus, thetransferred DNA is incorporated into the genome.

Attached to these pieces of targeting DNA are regions of DNA that mayinteract with the expression of a JNK activating phosphatase protein.For example, a promoter/enhancer element, a suppresser, or an exogenoustranscription modulatory element is inserted in the genome of theintended host cell in proximity and orientation sufficient to influencethe transcription of DNA encoding the desired JNK activating phosphataseprotein. The control element controls a portion of the DNA present inthe host cell genome. Thus, the expression of JNK activating phosphataseprotein may be achieved not by transfection of DNA that encodes the JNKactivating phosphatase gene itself, but rather by the use of targetingDNA (containing regions of homology with the endogenous gene ofinterest) coupled with DNA regulatory segments that provide theendogenous gene sequence with recognizable signals for transcription ofa JNK activating phosphatase protein.

In an exemplary method, expression of a desired targeted gene in a cell(i.e., a desired endogenous cellular gene) is altered by theintroduction, by homologous recombination into the cellular genome at apreselected site, of DNA which includes at least a regulatory sequence,an exon and a splice donor site. These components are introduced intothe chromosomal (genomic) DNA in such a manner that this, in effect,results in production of a new transcription unit (in which theregulatory sequence, the exon, and the splice donor site present in theDNA construct are operatively linked to the endogenous gene). As aresult of introduction of these components into the chromosomal DNA, theexpression of the desired endogenous gene is altered.

Altered gene expression, as used herein, encompasses activating (orcausing to be expressed) a gene which is normally silent (unexpressed)in the cell as obtained, increasing expression of a gene which mayinclude expressing a gene that is not expressed at physiologicallysignificant levels in the cell as obtained, changing the pattern ofregulation or induction such that it is different than occurs in thecell as obtained, and reducing (including eliminating) expression of agene which is expressed in the cell as obtained.

The present invention further relates to DNA constructs useful in themethod of altering expression of a target gene. In certain embodiments,the exemplary DNA constructs comprise: (a) a targeting sequence, (b) aregulatory sequence, (c) an exon, and (d) an unpaired splice-donor site.The targeting sequence in the DNA construct directs the integration ofelements (a)-(d) into a target gene in a cell such that the elements(b)-(d) are operatively linked to sequences of the endogenous targetgene. In another embodiment, the DNA constructs comprise: (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, (d) asplice-donor site, (e) an intron, and (f) a splice-acceptor site,wherein the targeting sequence directs the integration of elements(a)-(f) such that the elements of (b)-(f) are operatively linked to theendogenous gene. The targeting sequence is homologous to the preselectedsite in the cellular chromosomal DNA with which homologous recombinationis to occur. In the construct, the exon is generally 3′ of theregulatory sequence and the splice-donor site is 3′ of the exon.

If the sequence of a particular gene is known, such as the nucleic acidsequence of JNK activating phosphatase polypeptide presented herein, apiece of DNA that is complementary to a selected region of the gene canbe synthesized or otherwise obtained, such as by appropriate restrictionof the native DNA at specific recognition sites bounding the region ofinterest. This piece serves as a targeting sequence upon insertion intothe cell and will hybridize to its homologous region within the genome.If this hybridization occurs during DNA replication, this piece of DNA,and any additional sequence attached thereto, will act as an Okazakifragment and will be backstitched into the newly synthesized daughterstrand of DNA. The present invention, therefore, includes nucleotidesencoding a JNK activating phosphatase molecule, which nucleotides may beused as targeting sequences.

In vivo and in vitro gene therapy delivery of JNK activating phosphatasepolypeptide is also envisioned. In vivo gene therapy may be accomplishedby introducing the gene encoding JNK activating phosphatase polypeptideinto cells via local injection of a polynucleotide molecule or otherappropriate delivery vectors (Hefti, J. Neurobiology 25:1418-35 (1994)).For example, a polynucleotide molecule encoding JNK activatingphosphatase protein may be contained in an adeno-associated virus vectorfor delivery to the targeted cells (see, e.g., Johnson, PCT PublicationNo. WO 95/34670; PCT Application No. PCT/US95/07178). The recombinantadeno-associated virus (AAV) genome typically contains AAV invertedterminal repeats flanking a DNA sequence encoding JNK activatingphosphatase polypeptide operably linked to functional promoter andpolyadenylation sequences.

Alternative viral vectors include, but are not limited to, retrovirus,adenovirus, herpes simplex virus, and papilloma virus vectors. U.S. Pat.No. 5,672,344 describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5,399,346 provides examples of a process for providing a patient with atherapeutic protein by the delivery of human cells which have beentreated in vitro to insert a DNA segment encoding a therapeutic protein.Additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. No. 5,631,236 (involvingadenoviral vectors); U.S. Pat. No. 5,672,510 (involving retroviralvectors); and U.S. Pat. No. 5,635,399 (involving retroviral vectorsexpressing cytokines).

Nonviral delivery methods include liposome-mediated transfer, naked DNAdelivery (direct injection), receptor-mediated transfer (ligand-DNAcomplex), electroporation, calcium phosphate precipitation, andmicroparticle bombardment (e.g., gene gun). Gene therapy materials andmethods may also include inducible promoters, tissue-specificenhancer-promoters, DNA sequences designed for site-specificintegration, DNA sequences capable of providing a selective advantageover the parent cell, labels to identify transformed cells, negativeselection systems and expression control systems (safety measures),cell-specific binding agents (for cell targeting), cell-specificinternalization factors, transcription factors to enhance expression bya vector as well as methods of vector manufacture. Such additionalmethods and materials for the practice of gene therapy techniques aredescribed in U.S. Pat. No. 4,970,154 (electroporation techniques); PCTApplication No. WO 96/40958 (nuclear ligands); U.S. Pat. No. 5,679,559(concerning a lipoprotein-containing system for gene delivery); U.S.Pat. No. 5,676,954 (involving liposome carriers); U.S. Pat. No.5,593,875 (concerning methods for calcium phosphate transfection); andU.S. Pat. No. 4,945,050 (wherein biologically active particles arepropelled at cells at a speed whereby the particles penetrate thesurface of the cells and become incorporated into the interior of thecells). Expression control techniques include chemical inducedregulation (see, e.g., PCT Application Nos. WO 96/41865 and WO97/31899), the use of a progesterone antagonist in a modified steroidhormone receptor system (see, e.g., U.S. Pat. No. 5,364,791), ecdysonecontrol systems (see, e.g., PCT Application No. WO 96/37609), andpositive tetracycline-controllable transactivators (see, e.g., U.S. Pat.No. 5,589,362; U.S. Pat. No. 5,650,298; and U.S. Pat. No. 5,654,168).

One manner in which gene therapy can be applied is to use the JNKactivating phosphatase gene (either genomic DNA, cDNA, and/or syntheticDNA encoding a JNK activating phosphatase polypeptide, or a fragment,variant, or derivative thereof) which may be operably linked to aconstitutive or inducible promoter to form a “gene therapy DNAconstruct.” The promoter may be homologous or heterologous to theendogenous JNK activating phosphatase gene, provided that it is activein the cell or tissue type into which the construct will be inserted.Other components of the gene therapy DNA construct may optionallyinclude: DNA molecules designed for site-specific integration (e.g.,endogenous flanking sequences useful for homologous recombination), atissue-specific promoter, enhancers, silencers, DNA molecules capable ofproviding a selective advantage over the parent cell, DNA moleculesuseful as labels to identify transformed cells, negative selectionsystems, cell specific binding agents (as, for example, for celltargeting), cell-specific internalization factors, and transcriptionfactors to enhance expression by a vector as well as factors to enablevector manufacture.

This gene therapy DNA construct can then be introduced into thepatient's cells (either ex vivo or in vivo). One means for introducingthe gene therapy DNA construct is via viral vectors. Suitable viralvectors typically used in gene therapy for delivery of gene therapy DNAconstructs include, without limitation, adenovirus, adeno-associatedvirus, herpes simplex virus, lentivirus, papilloma virus, and retrovirusvectors. Some of these vectors, such as retroviral vectors, will deliverthe gene therapy DNA construct to the chromosomal DNA of the patient'scells, and the gene therapy DNA construct can integrate into thechromosomal DNA; other vectors will function as episomes and the genetherapy DNA construct will remain in the cytoplasm.

Another means to increase endogenous JNK activating phosphatasepolypeptide expression in a cell via gene therapy is to insert one ormore enhancer elements into the JNK activating phosphatase polypeptidepromoter, where the enhancer elements can serve to increasetranscriptional activity of the JNK activating phosphatase polypeptidegene. The enhancer elements used will be selected based on the tissue inwhich one desires to activate the gene-enhancer elements known toconfer-promoter activation in that tissue will be selected. For example,if a gene encoding an JNK activating phosphatase polypeptide is to be“turned on” in T-cells, the Ick promoter enhancer element may be used.Here, the functional portion of the transcriptional element to be addedmay be inserted into a fragment of DNA containing the JNK activatingphosphatase polypeptide promoter (and optionally, inserted into a vectorand/or 5′ and/or 3′ flanking sequences, etc.) using standard cloningtechniques. This construct, known as a “homologous recombinationconstruct,” can then be introduced into the desired cells either ex vivoor in vivo.

Gene therapy can be used to decrease JNK activating phosphatasepolypeptide expression by modifying the nucleotide sequence of theendogenous promoter. Such modification is typically accomplished viahomologous recombination methods. For example, a DNA molecule containingall or a portion of the promoter of the JNK activating phosphatase geneselected for inactivation can be engineered to remove and/or replacepieces of the promoter that regulate transcription. Here, for example,the TATA box and/or the binding site of a transcriptional activator ofthe promoter may be deleted using standard molecular biology techniques;such deletion can inhibit promoter activity thereby repressingtranscription of the corresponding JNK activating phosphatase gene.Deletion of the TATA box or transcription activator binding site in thepromoter may be accomplished by generating a DNA construct comprisingall or the relevant portion of the JNK activating phosphatasepolypeptide promoter (from the same or a related species as the JNKactivating phosphatase gene to be regulated) in which one or more of theTATA box and/or transcriptional activator binding site nucleotides aremutated via substitution, deletion and/or insertion of one or morenucleotides such that the TATA box and/or activator binding site hasdecreased activity or is rendered completely inactive. This construct,which also will typically contain at least about 500 bases of DNA thatcorrespond to the native (endogenous) 5′ and 3′ DNA sequences adjacentto the promoter segment that has been modified, may be introduced intothe appropriate cells (either ex vivo or in vivo) either directly or viaa viral vector as described above. Typically, integration of theconstruct into the genomic DNA of the cells will be via homologousrecombination, where the 5′ and 3′ DNA sequences in the promoterconstruct can serve to help integrate the modified promoter region viahybridization to the endogenous chromosomal DNA.

Other gene therapy methods may also be employed where it is desirable toinhibit the activity of one or more JNK activating phosphatasepolypeptides. For example, antisense DNA or RNA molecules, which have asequence that is complementary to at least a portion of the selected JNKactivating phosphatase polypeptide gene can be introduced into the cell.Typically, each such antisense molecule will be complementary to thestart site (5′ end) of each selected JNK activating phosphatase gene.When the antisense molecule then hybridizes to the corresponding JNKactivating phosphatase mRNA, translation of this mRNA is prevented.

Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more JNK activating phosphatasepolypeptides. In this situation, the DNA encoding a mutant full lengthor truncated polypeptide of each selected JNK activating phosphatasepolypeptide can be prepared and introduced into the cells of a patientusing either viral or non-viral methods as described above. Each suchmutant is typically designed to compete with endogenous polypeptide inits biological role.

Uses of JNK Activating Phosphatase Nucleic Acids and Polypeptides

Nucleic acid molecules of the invention may be used to map the locationsof the JNK activating phosphatase gene and related genes on chromosomes.Mapping may be done by techniques known in the art, such as PCRamplification and in situ hybridization.

The nucleic acid molecules are also used as anti-sense inhibitors of JNKactivating phosphatase polypeptide expression. Such inhibition may beeffected by nucleic acid molecules that are complementary to andhybridize to expression control sequences (triple helix formation) or toJNK activating phosphatase mRNA. Anti-sense probes may be designed byavailable techniques using the sequence of the JNK activatingphosphatase genes disclosed herein. Anti-sense inhibitors provideinformation relating to the decrease or absence of an JNK activatingphosphatase polypeptide in a cell or organism.

Hybridization probes may be prepared using an JNK activating phosphatasegene sequence as provided herein to screen cDNA, genomic or syntheticDNA libraries for related sequences. Regions of the DNA and/or aminoacid sequence of JNK activating phosphatase polypeptide that exhibitsignificant identity to known sequences are readily determined usingsequence alignment algorithms disclosed above and those regions may beused to design probes for screening.

The nucleic acid molecules of the invention may be used for genetherapy. Nucleic acid molecules that express JNK activating phosphatasepolypeptide in vivo provide information relating to the effects of thepolypeptide in cells or organisms.

JNK activating phosphatase nucleic acid molecules, fragments, variants,and/or derivatives that do not themselves encode biologically activepolypeptides may be useful as hybridization probes in diagnostic assaysto test, either qualitatively or quantitatively, for the presence of JNKactivating phosphatase DNA or corresponding RNA in mammalian tissue orbodily fluid samples.

JNK activating phosphatase polypeptides, fragments, variants, and/orderivatives may be used to screen agents for preventing, treating ordiagnosing JNK-mediated disorders.

JNK activating phosphatase polypeptides, fragments, variants, and/orderivatives, whether biologically active or not, are useful forpreparing antibodies that bind to an JNK activating phosphatasepolypeptide. The antibodies may be used for in vivo and in vitrodiagnostic purposes, including, but not limited to, use in labeled formto detect the presence of JNK activating phosphatase polypeptide in abody fluid or cell sample. The antibodies may also be used to prevent ortreat JNK-mediated disorders. The antibodies may bind to an JNKactivating phosphatase polypeptide so as to diminish or block at leastone activity characteristic of an JNK activating phosphatasepolypeptide, or may bind to a polypeptide to increase an activity.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

EXAMPLE 1 Cloning of Mouse JNK Activating Phosphatase Polypetptide Gene

Materials and methods for cDNA cloning and analysis are described inSambrook et al. supra.

Original sequence tags identified as “LS20-1” and “LS20-2” came from asequence survey of several hundred differential display PCR (DD-PCR)clones. The DD-PCR inserts were subcloned into the plasmid vector pGEM-T(Promega). The common insert in tag LS20-1 and LS20-2 exhibited nosequence homologies with other known genes, expressed sequence tags(ESTs), or sequence-tagged sites (STSs) in a contemporaneous search ofGenBank. The insert in LS20-1 was in a sense orientation with respect tothe T7 polymerase transcription initiation site in the pGEM vector. Theinsert in LS20-2 was in an antisense orientation with respect to thissame promoter (orientation was deduced by labelling probes in bothorientations and hybridizing to Northern blots). The clone LS20-2, whendigested with the restriction endonuclease PstI yields a good templatefor the synthesis of a radio-labelled riboprobe using the T7transcription initiation site. Such a riboprobe template was used forsubsequent screening of lambda phage libraries for full length LS20cDNA.

Northern analysis of LS 20 expression in the mouse demonstrated goodlevels of mRNA in the skeletal muscle, among other tissues. Skeletalmuscle was chosen as the target source because we had several “good”mouse and human skeletal muscle cDNA libraries (Clontech and Stratagene)in hand.

A mouse skeletal muscle cDNA library (Clontech) was titered. One millionplacques were plated and lifted in duplicate onto nitrocellulose filters(Schleicher and Schuell, Keens, N.H.). Hybridization with the LS20riboprobe was performed in Stark's buffer (50% formamide; 50 mMpotassium phosphate, pH 6.5; 5×SSC; 1% SDS; 5× Denhardt's; 0.05% sodiumsarcosyl; and 300 μg/mL salmon sperm DNA) at 42° C., overnight (ON).Filters were washed to a final stringency of 1×SSC, 0.1% SDS, 42° C.,and exposed to X-ray film (X-OMAT AR, Eastman Kodak, Rochester, N.Y.) ONat −70° C. with intensifying screens. Films were developed and 13 plaquepools (from plates 2, 7, 8, 9, 10 (2ea.), 15, 16, 17, 18 (2ea.), 19, and20) hybridizing on both pairs of duplicate lifts were identified andisolated.

To facilitate rapid isolation of novel sequence extending the known LS20sequence, anchored PCR was used on these 13 primary plaque pools. Threesynthetic oligonucleotides were prepared. The first oligonucleotide,1065-30 (SEQ ID NO: 17), being identical in sequence to a 32 nucleotideregion of the left lambda phage vector arm and flanking on one side theinsert cloning site within the vector: (SEQ ID NO:17)(5′-CCTTTTTGAGCAAGTTCAGCCTGGTTAAGTCC-3′).

The second oligonucleotide, 1386-5 8 (SEQ ID NO: 18), being identical insequence to a 33 nucleotide string near the 5′-end of the LS20-2 insert:(SEQ ID NO:18) (5′-GGAGGCCTCTCTCTGTGTGTGTGGAGCCCTCAGG-3′);

The third oligonucleotide, 1386-59 (SEQ ID NO: 19), being complementary(anti-sense) to a 31 nucleotide strings near the 3′-end of the LS20-2insert: (SEQ ID NO:19) (5′-GGCAGCACCAGCCTGAACTTTGCAATATTTC-3′).The lambda phage-specific primer was combined with either of the twoLS20-specific primers and the PCR was performed. Four of the originalplaque pools allowed PCR amplification with one, but not the other, ofthe LS20-specific primers (pools 7, 17, 18, and 20). The PCR productsthus amplified were gel purified and named #s 3, 18, 1 9, and 26,respectively (number designation corresponding to gel lane). Gelpurified fragments were subcloned into the plasmid vector pCR2.1(InVitrogen, Inc., San Diego, Calif.) and individual clones isolated(clones 3-2, 19-27, and 26-31).

Inserts from these three plasmid clones were sequenced on both strandsand compared to the original LS20 sequence. The longest 5-extension ofthe original LS20 sequence was contained in the clone 26-31, but did notextend into LS20 coding region. A 1.1 Kbp probe template was prepared bydigesting the clone 26-31 with the restriction enzyme NcoI. This probetemplate was used to synthesize random hexamer-primed probes for furtherscreening of a Stratagene mouse skeletal muscle library.

A second mouse skeletal muscle library (Stratagene) was plated andlifted as before, and probed with a random hexamer-primed probe preparedfrom the 1.1 Kbp LS20 template described above. The filters werehybridized as for the first library, then washed to a final stringencyof 2×SSC, 0.1% SDS, 42C, 5 m. Upon exposure of films, 20 primarypositive plaque pools were isolated (pools 2-1, 2-2, 2-3, 3-1, 6-1, 6-2,6-3, 7-1, 12-1, 12-2, 12-3, 16-1, 16-2, 16-3, 16-4, 16-5, 16-6, 18-1,18-2, 18-3). Anchored PCR was performed as described above, using thesame primers. Primary plaque pools 2-3, 16-2, 164, and 18-4 all yieldedPCR products with the LS20-specific primer 1386-58, but not 1386-59. Allpools except 2-3 allowed amplification of multiple bands, therefore thesingle amplified band from pool 2-3 was gel purified, subcloned(pCR2.1), and sequenced. The sequence of subclone 2-3b (via PCRamplification of primary pool 2-3) was obtained by us and shown tocontain an open reading frame encoding a putative protein with homologyto a class of proteins known as dual-specificity phosphatases.Subsequent cloning of the human orthologue of this cDNA (describedbelow) demonstrated that the mouse LS20 cDNA was full length and didencode the bona fide initiation methionine for the protein known here asJKAP or JNK activating phosphatase.

EXAMPLE 2 Cloning of Human JNK Activating Phosphatase Polypeptide Gene

A FASTA search of Genbank EST sequences with the novel mouse LS20 cDNAsequence revealed a high homology hit with a human EST designated clone249002. The clone 249002 was purchased from an IMAGE consortium supplier(Genome Systems, St. Louis, Mo.) and sequenced in its entirety. Theinsert was short, 614 bp. The insert of clone 249002 was isolated bydigestion with the restriction enzymes EcoRI and NotI, gel-purified andused as a template for the synthesis of random hexamer-primed probesused in subsequent screens of a human fetal liver cDNA library(Clontech). In addition, two new oligonucleotide primers weresynthesized based on the human LS20 sequence. Sense (1470-25) (SEQ IDNO: 20) and anti-sense (1470-26) (SEQ ID NO: 21) primers were designedto allow amplification of a 143bp internal fragment of the human LS20sequence or for use in an anchored PCR scheme similar to that used inthe cloning of the mouse LS20 cDNA described above. (5′-C AGCAGCGGATTCACCATC-3′) (SEQ ID NO: 20) (5′-GCGATCACCAGTGTCACGC-3′) (SEQ ID NO:21)

A human fetal liver cDNA library was plated and lifted in duplicate, asdescribed above. A random hexamer-primed probe from the ca.600bp 249002template was hybridized with these filters at 42C, O/N. The filters werewashed to a final stringency of 0.2×SSC, 0.1% SDS, 42C and exposed tofilm. Eight primary positive pools were identified and isolated (pools5-1, 5-2, 10-1, 16-1, 16-2, 16-3, 164, 16-5). Anchored PCR was performedon these pools, using the vector-specific primer 1065-30 and one of thetwo hLS20-specific primers 1470-25 or -26. A PCR product from one ofthese primary pools (16-1A) was gel purified, subcloned and sequenced.

A sequence from 16-1A-derived subclones was obtained. The sequenceencoded a full length human LS20 cDNA, complete with upstream stop codonin-frame with the predicted initiation methionine, the full orf, andtermination codon. DNA sequencing was performed on both strands of eachtemplate using the Taq Dye Terminator Cycle Sequencing kit (AppliedBiosystems, Foster City, Calif.) and primers appropriate to the cloningvector on an automated DNA sequencer (Model 377, Applied Biosystems), asper the manufacturer's recommendations.

EXAMPLE 3 The JKAP Amino Acid Sequence Analysis and Expression Pattern

JKAP was initially identified in a screen designed to isolatetranscripts differentially upregulated in adult mouse bone marrowhematopoietic cells, FACS-sorted Lin⁻ Sca-1⁺ subset of whole mouse bonemarrow cells derived from C57816 strain mice. Differential-display PCR(DDPCR) (DDPCR was performed as described (R. Jurecic, T. Nguyen, J. W.Belmont, Trends Genet. 12, 502 (1996); R. Jurecic, R. G. Nachtman, S. M.Colicos, J. W. Belmont, Anal. Biochem. 259, 235 (1998)]) was employed toidentify the JKAP transcript, among others, as being expressedpreferentially in the stem-cell-enriched Lin⁻ Sca-1⁺ population of mousebone marrow cells obtained by FACS, compared to the Lin⁻ Sca-1⁻population which is deficient in stem-cell activity. JKAP mapped tomouse proximal Chromosome 13 by backcross panels (add marker order]. Thefull-length JKAP cDNA consisted of 3012 bp (cDNA cloning—A full-lengthcDNA clone of JKAP was isolated from a skeletal muscle library, based onpreliminary expression data), and contained a 700 bp open reading framewith high similarity to a subgroup of protein tyrosine phosphatases, thedual-specificity phosphatases, that have activity in the MAPK pathways(FIG. 3A). The predicted JKAP protein contains the catalytic C-terminaldomain of dual-specificity phosphatases, but lacks an N-terminalnon-catalytic domain, and is consequently ˜100 aa shorter than mostother MAPK phosphatases. All residues of the signature motifI/VHCxxGxSRS of dual-specificity phosphatases (J. M. Denu, J. A.Stuckey, M. A. Saper, J. E. Dixon, Cell 87, 361 (1996); N. K. Tonks andB. G. Neel, ibid, p. 365) are conserved in the JKAP phosphatase. Maximumparsimony analysis restricted to the catalytic domain indicates thatIKAP is not an orthologue of previously described MAPK phosphatases(FIG. 3B). By Northern blot analysis, JKAP is expressed in most adultmouse tissues examined (FIG. 3C). The detection of two transcripts, thepredominant of which corresponds with the full-length JKAP cDNA,suggests tissue-specific splicing or processing. Either or both the 3.0kb transcript and the 1.3 kb transcript were present in brain, testis,heart, liver, and kidney, and at lower levels in skeletal muscle.

EXAMPLE 4 In Situ Hybridization of Mouse Bone Marrow Cells

To further characterize the expression pattern of JKAP in mouse tissues,an in situ hybridization analysis was conducted. Hybridization of LinSca-1⁺ and Lin Sca-1⁻ cells from adult mouse bone marrow confirmeddifferential expression of JKAP (FIGS. 4, A and B), suggesting a rolefor JKAP unique to cells within this hematopoietic precursor population.Lin Sca-1⁺ and Lin Sca-1⁻ cells obtained through FACS from mouse bonemarrow were resuspended and fixed in 4% paraformaldehyde in PBS, thendeposited on RNase-free, TESPA (Sigma)—treated glass slides. In situhybridization was conducted as described [U. Albrecht, G. Eichele, J. A.Helms, H. C. Lu, in Molecular and Cellular Methods in DevelopmentalToxicology, G. P. Daston, Ed. (CRC Press, Boca Raton, 1997), pp. 23-48],with modifications made for cells. 0.5-1 μg/nL digoxigenin-labelled JKAPriboprobe, corresponding to nucleotide positions 2251 to 2566 of theJKAP cDNA, was hybridized to the cells without coverslipping asdescribed [W. Nurnberg, B. M. Czarnetzki, D. Schadendorf, Biotechniques18, 406 (1995)].

Lin⁻ Sca-1⁺ and Lin⁻ Sca-1⁻ mouse cells obtained through flow cytometrywere resuspended and fixed in 4% paraformaldehyde in PBS, then depositedon RNase-free, TESPA (Sigma)-treated glass slides. The cells wereair-dried at room temperature for 20-30 min, rinsed in PBS for 5 min,acetylated in 0.1 M triethanolamine-HCl (Fisher) pH 8.0 with 0.72%acetic anhydride (Fisher) for 10 min, and rinsed in PBS for 5 min. Thecells were then equilibrated in 0.9% NaCl for 5 min, carried through adehydration series of 30%, 50%, 70%, 80%, 95%, and 100% EtOH, andfinally air-dried for 20 min at room temperature. The cells wereencircled using a hydrophobic pen (Electronic Microscopy Sciences) andincubated with 50 μL of prehybridization solution (50% formamide, 20 mMtris-HCl pH 8.0, 0.3 mM NaCl, 10% dextran sulfate, 1× Denhardt's, 0.5mg/mL yeast tRNA, 10 mM DTM) at 50-55 C for 1 hr in a humid chamber.Digoxigenin-labelled riboprobe in 50 μL hybridization solution, heatedat 95 C for 5 min, was added to a final concentration of 50-100 ng per100 μL total hybridization volume, and incubated at 50-55 C for 14-18hrs. Post-hybridization, the slides were washed with constant agitationin 2×SSC, 0.1% Tween-20 at 55-60° C. for 1 hr, 0.5×SSC, 0.1% Tween-20 at37° C. for 30 min, and 0.5×SSC, 0.1% Tween-20 at room temperature for 30min. For antibody detection of the DIG-labelled riboprobe, the cellswere rinsed in PBS, incubated in blocking buffer (10% lamb senum in PBS0.1% Tween-20) at 37° C. for 30 min, then incubated with sheepanti-DIG-APconjugated antibody (Boehringer Mannheim) diluted 1:500 inblocking buffer at 37° for 1 hr. Following antibody incubation, theslides were rinsed in PBS, 0.1% Tween-20, washed in PBS, 0.1% Tween-20at room temperature for 10 min with constant agitation, then washed indetection buffer (100 mM tris-HCl pH 9.5, 100 mM NaCl, 50 mM MgCl₂) atroom temperature for 10 min with constant agitation. For calorimetricdetection of the antibody, the slides were incubated in substratesolution (450 μg/mL nitro blue tetrazolium chloride(NBT), 175 /mL5-bromo-4-chloro-3-indolyl-phosphate, 4-toluidine salt (BCIP, 240 μg/mLlevarnisole in detection buffer) at room temperature for 30-60 min in adark, humid chamber. Finally, the reaction was terminated by rinsing theslides thoroughly in water, and coverslips were mounted using an aqueousmounting medium.

EXAMPLE 5 In Situ Hybridization of E10.5 Mouse Embryos

Whole-mount in situ hybridization of embryonic day (E) 10.5 mouseembryos detected highest levels of JKAP transcripts in the somites andbranchial arches (FIGS. 4, C through E). Whole mount in situhybridization to E10.5 mouse embryos was performed as described [D. G.Wilkinson, in In situ Hybridization: A Practical Approach, D. G.Wilkinson, Ed. (IRL Press, Oxford, 1992), pp. 75-83], with the exceptionthat hybridization was carried out in stationary tubes, using 200 ng/mLof digoxigenin-labelled riboprobes corresponding to nucleotide positions17 to 1522 of the JKAP cDNA. On day 1, embryos (fixed overnight in 4%paraformaldehyde in PBT (PBS+0.1% Tween-20) washed three times in PBTand stored in 100% methanol until the day of hybridization) wererehydrated through a 75% -50%-25%-0% methanol series, washed twice inPBT, and incubated for 15 minutes in a solution of 10 μg/ml proteinase Kin PBT. The proteinase K reaction was stopped by washing in 2 mg/mlglycine in PBT, followed by refixation in 4% paraformaldehyde/0.2%glutaraldehyde in PBT. Embryos were washed three times in PBT,prehybridized for one hour at 65° C. in hybridization solution (5×SSC,50% formamide, 1% SDS, and 100 μg/ml each of heparin and yeast tRNA),and hybridized overnight in fresh hybridization solution to which 100ng/ml digoxygenin-labelled riboprobe had been added. On day two, thefollowing washes were performed: (1) twice for 30 minutes in WashSolution I (5×SSC, 50% formamide, 1% SDS) at 65° C.; (2) once for 10minutes in 1:1 Wash Solution I:Wash Solution II (0.5M NaCl, 10 mMTris-HCl pH 7.5, 0.1% Tween-20) at 65° C.; (3) three times for 5 minutesin Wash Solution II at room temperature; (4) once for 30 minutes in WashSolution II+100 μg/ml RNase A at 37° C.; (5) once for 5 minutes in WashSolution II; (6) twice for 30 minutes in Wash Solution III (2×SSC, 50%formamide, 0.1% Tween-20) at 65° C.; (6) three times for 5 minutes inTBST (140 mM NaCl, 2.7 mM KCl, 25 mM Tris-HCl pH 7.5, 0.1% Tween-20);(7) once for 90 minutes in TBST+10% normal lamb serum; (8) overnight inTBST+1:2000 dilution of sheep and digoxygenin Fab fragments in 4° C.(Fab fragments preabsorbed for 60 minutes to heat-inactivated E14.5mouse embryo powder in TBST+1% normal lamb serum). On day three, theembryos were washed five times for 60 minutes in TBST at roomtemperature, followed by a sixth wash overnight at 4° C. On day four,the embryos were washed twice for 10 minutes in CT solution (100 mMTris-HCl pH 9.5, 150 mM NaCl, 25 mM MgCl₂, 2 mM levamisole, 0.1%Tween-20), and then incubated in the dark at room temperature in CTsolution+337.5 μg/ml nitroblue tetrazolium salt+175 μg/ml5-bromo4chloro-3-indolyl-phosphate+10% polyvinyl alcohol (Barth andIvarie, 1994). After approximately five to six hours of colordevelopment, embryos were washed three times in PBT, cleared in 50%glycerol in PBT, followed by storage at 4° C. in 50% glycerol in PBT.Embryos were embedded in paraffin and sectioned according to Albrecht etal. (199A). Sections were counterstained with nuclear fast red.

Color development was enhanced by the addition of polyvinyl alcohol asdescribed [J. Barth and R. Ivarie, Biotechniques 17, 324 (1994)]. Noexpression was observed in the embryonic dorsal aorta, a regionidentified as a site of definitive hematopoieses in chick and humanembryos at similar developmental stages (T. Jaffredo, R. Gautier, A.Eichmann, F. Dieterlen-Lievre, Development 125, 4575 (1998); M.Labastie, F. Cortes, P. Romeo, C. Dulac, B. Peault, Blood 92, 3624(1998)); indicating that the earliest intraembryonic hematopoietic cellsdo not express JKAP (FIG. 4F).

EXAMPLE 6 Transfection of 293T Cells

Given the close sequence similarity of JKAP to MAPK phosphatases, theactivity of JKAP phosphatase in MAPK cascades was examined. We assayedMAPK activity in 293T cells co-transfected with the JKAP phosphatase andeither JNK1, ERK2, or p38 using an immunocomplex kinase assay.Endogenous JNK1 and over-expressed HA-JNK1 were immunoprecipated byincubation with rabbit anti-JNK1 polyclonal antibody (Ab101) and mouseanti-HA monoclonal antibody (12CAS), respectively, plus proteinA-agarose beads (Bio-Rad) in lysis buffer (20 mM HEPES, pH 7.4, 2 mMEGTA, 50 mM glycerophosphate, 1% Triton X-100, 10% glycerol, 1 mMdithiothreitol, 2 μg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride. 1mM NaCL and 1 mM Na₃ VO₄). The precipitates were washed twice with lysisbuffer, twice with LiCl buffer (500 nM LiCl, 100 mM Tris-HCl, pH 7.6 and0.1% Triton X-100), and twice with kinase buffer (500 mM LiCl, 100 mMTris-HCl, pH 7.6, and 0.1% Triton X-100), and twice with kinase buffer(20 mM MOPS, pH 7.2, 2 mM EGTA, 10 mM MgCl₂, 1 mM dithiothrreitol, 0.1%Triton X-100, and 1 mN Na₃VO₄). The pellets were then mixed with 1 μg ofGST-clun (1-79), 15 μM ATP, and 10 μCi of [γ⁻³²P] ATP in 30 μL of kinasebuffer. The kinase reaction was performed at 30 C for 30 min. andterminated with an equal volume of SDS sampling buffer. The reactionmixtures were analyzed by SDS-PAGE and autocadiography. Polyclonal Ab101was derived from rabbits that were immunized with peptide N′ - - -CKNGVIRGQPSPLAQVQ (SEQ ID NO:23) Q - - - C′.The carrier used was KLH. This antibody was prepared by standard methodsusing two injections and termination three weeks after burst injection.One suitable source for preparation of Ab101 (titer>1:10 K) is GenemedSynthesis, Inc See also Chen, Y.-R, Meyer, C. F., and Tan, T.-H.,(1996). Persistent activation of c-Jun N-terminal kinase 1 (JNK1) ingamma radiation-induced apoptosis. J.Biol. Chem. 271:631-634; Hu, M.C.-T., Qiu, W. R, Wang, X., Meyer, C. F, and Tan, T.-H., (1996). HumanHPK1, a novel human hematopoietic projenitor kinase that activates theJNK/SAPK kinase cascade. Gene & Development 10:2251-2264.; Wang W.,Zhou, G., Hu, M. C.-T., Yao, Z., and Tan,T.-H., (1999). Activation ofHematopoietic progenitor kinase 1 (HPK1)-dependent, stress-activatedc-Jun N-terminal kinase (JNK) pathway by transforming growth factor beta(TGF-beta)-activated kinase (TAK1), a kinase mediator of TGF-beta signaltransduction. J. Biol. Chem. 272:22771-22776; Ensenat, D., Yao Z., WangX. S., Kori, R., Zhou, G., Lee, S. C, and Tan, T.-H., (1 999). A novelSrc homology 3 domain-containing adaptor protein, HIP55, that interactswith hematopoietic progenitor kinase 1. J. Biol. Chem. 274:33945-33950;Zhou, G., Lee, S. C, Yao, Z., and Tan,T.-H., (1999). Hematopoieticprogenitor kinase 1 is a component of transforming growth factorbeta-induced c-Jun N-terminal kinase signaling. J. Biol. Chem.274:13133-13138.

Unexpectedly, JKAP activated JNK1 but did not activate ERK2 or p38 (FIG.5). Transient transfections were performed in 293T cells by using thecalcium phosphate precipitation protocol provided by Specialty Media,Inc., Lavallette, N.J. Briefly, 293T cells were plated at a density of1.5×10⁵ cells/35 mm plate well and transfected with the indicatedamounts of various DNA plasmids the next day. Empty vectors were used tonormalize the amount of transfected DNA. The plasmid encodingFgalactosidase was cotransfected into the cells as an internal controlto monitor the transfection efficiency.

Transfection with a mutant, inactive form of JKAP (JKAP-C88S) did notresult in JNK activation, demonstrating that this response was dependenton intact. JKAP phosphatase activity. These. data suggest a specificrole for JKAP in the JNK pathway. The mutant JKAP-C88S differs fromwild-type JKAP in a single amino acid substitution of serine forcysteine at position 88. No other regions of the molecule are altered.The cysteine at position 88 is the central catalytically active residein the nucleophilic attack of the phosphatase on the substrate. Thus,substitution of a serine at this position renders the JKAP moleculebiochemically inactive.

TNF-alpha induced JNK activity was blocked by mutant JKAP. 293T cellstransfected with 0.1 ug of HA-JNK1 alone or HA-JNK1 plus 2 ug ofJKAP-C88S were treated with TNF-alpha (10 ng/ml). After 30 minutes,cells were collected and cell lysates prepared. HA-JNK1 wasimmunoprecipitated with an anti-HA antibody (12CA5), and immunocomplexkinase assays were performed using GST-cJun (1-79) as a substrate.Equivalent levels of HA-JNK1 expression were verified by immunoblot (IB)analysis using anti-HA (12CA5).

EXAMPLE 7 Effect of JKAP on Regulating JNK Activation

To determine whether JKAP is necessary for JNK activation, the responseof cells deficient in the JKAP phosphatase to various stimuli wastested. Mouse embryonic stem (ES) cells heterozygous for the deletion ofJKAP through homologous recombination were generated, and homozygousclones were derived by secondary selection (FIG. 6). JKAP^(+/+) andJKAP^(−/−) cells were exposed to one of several cytokines orenvironmental stresses (FIG. 7). JNK targeted ES cell clones weregenerated essentially as described [A. Bradley, pers. comm., and inTeratocarinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, Ed. (IRL Press. Oxford, 1987, pp. 113-152.] Briefly, atargeting vector was designed that deletes the two coding exons of JKAPwhich encode the catalytic domain of the phosphatase. The vector wasconstructed using DNA from a mouse 129/SV/EV total genomic DNA library.This vector was electroporated into an AB2.2 ES cell library from129/SV/EV mice library. The AB2.2 ES cell lines were derived from BlackAgouti 129 mice. This cell line is an XU cell line that when grown onSNL76/7 feeder cells are predominantly normal. SNL76/7, a clonal isolateof STO cells that have been transfected with a LlF expression cassetteand RV4.0 (a neo expression cassette, have been selected for theirability to maintain ES cells in a “normal state”. Resulting clones werescreened by Southern blot analysis for evidence of homologousrecombination. Two targeted clones were microinjected into E3.5 C57BU61blastocysts, at approximately 20 ES cells per blastocyst

˜80% confluent JKA^(≠) and JKAP⁺ES cells in 60 mm dishes were treatedwith TNF-α (10 ng/mL), TGF-β (10 ng/mL), and IL-1 (10 ng/mL) for 10min., and sorbitol (400 nM), and UV-C (300 J/m²) for 30 min. Cells werethen collected and cell lysates prepared. Endogenous JNK1 wasimmunoprecipitated with an anti-JNK1 (Ab101), and immunocomplex kinaseassays were performed using GST-cJun (1-79) as a substrate. Theexpression levels of JNK1 in JKAP^(≠), JKAP⁺ES cells were monitored byimmunoblot analysis using anti-JNK1 antibody (Ab101).Stimulation by theproinflammatory cytokines tumor necrosis factor (TNF)-α and transforminggrowth factor (TGF)-β which specifically engage the JNK pathway (A. J.Flint, T. Tiganis, D. Barford, N. K. Tonks, Proc. Natl. Acad. Sci. 941680 (1997); A. J. Garton, A. J. Flint, N. K. Tonks, Mol. Cell Biol. 166408 (1996)), resulted in greatly reduced JNK activation in JKAP^(−/−)cells. Stimulation by interleukin (IL)-1 or hyperosmolar shock alsoreduced JNK activation, while stimulation by UV radiation resulted inonly a mild reduction of JNK activation in JKAP^(−/−) cells. These datademonstrate the requirement of JKAP for full induction of JNK activityunder stimulus induction.

Cultures of fibroblasts derived from JKAP+/+ and JKAP−/− embryos werestimulated with 100 ng/ml 4-alpha-phorbol 12-myristate 13-acetate (PMA).After various times cell lysates were prepared and immunoprecipitatedwith anti-ERK2. ERK2 kinase activity was measured by phosphorylation ofMBP. MBP was separated on PAGE gel and the phosphorylated productmeasured by phsphorimager analysis. See FIG. 7(g). Cultures offibroblasts derived from JKAP+/+ and JKAP−/− embryos were stimulatedwith UV-C (100 J/m2) and at various times thereafter, immunocomplexassays for p38 activity were performed. Endogenous p38 wasimmunoprecipitated and then its kinase activity was measured on eitherMBP or GST-ATF2 with similar results. See FIG. 7(h). Compared towild-type cells, JKAP−/− embryonic fibroblasts respond to PMA withactiviation of the ERK2 pathway and respond to UV-C stimulation withactiviation of the p38 stress response pathway. These results indicatethat the main activity of JKAP in these cells is the JNK pathway.Pathway specificity may be an advantage for a compound that specificallyenhance or interfere with JKAP activity and is not a prerequisite forJKAP utility.

EXAMPLE 8 Production of JNK Activating Phosphatase Polypeptide inMammalian Cells

To express human JNK activating phosphatase in vitro, cDNA encodinghuman LS20 was subcloned into retroviral construct MSCV2.1 (Clontech).After verifying the sequence of LS20 insert, this expression vector wastransfected into GP+E86 packaging cell line (Genetix Pharmaceuticals,Cambridge, Mass.) and supernatant containing recombinant virus wasproduced. This supernatant was then used to infect mouse NIH-3T3 cells(American Type Culture Collection) and infected clones were selectedwith G418 (Gibco BRL). Extracts from either control cells or cellsinfected with recombinant retrovirus containing LS20 cDNA were generatedusing lysis buffer (PBS+0.5% Nonidet P20 (Sigma, St. Louis, Mo.)). 30 μLof extracts were run on a 10% Tris-glycine gel (Novex, San Diego,Calif.). Proteins were then transferred to nitrocellulose paper(Schleicher and Schuell) and were blocked in TBS buffer (20 mM Tris, pH7.5, 138 mM NaCl, 0.1% Tween 20 (Boehringer Mannheim)) with 5% dry milk.Anti-LS20 antiserum from animal #1436 was used in western blot analysisto detect LS20 at a ratio of 1:1000 in TBS buffer. Anti-rabbit Igantibody #NA 934 (Amersham Pharmacia Biotech, Rahway, N.J.) conjugatedwith horse radish peroxidase was used for the secondary detection at aratio of 1:2000. Detection of LS20 positive bands were performed withEnhanced Chemiluminacence (ECL) kit (Amersham, Piscataway, N.J.) usingmanufacturer's recommended protocol. Two bands, one 26 kiloDalton (kD)and the other 20 kD, were detected in the extract from cells infectedwith LS20 recombinant retrovirus but not in the control extract. Thenature of the cause of double bands is not known.

EXAMPLE 9 Production of Anti-JNK Activating Phosphatase PolypeptideAntibodies

Antibodies to JNK activating phosphatase polypeptides may be obtained byimmunization with purified protein or with JNK activating phosphatasepeptides produced by biological or chemical synthesis. Procedures forgenerating antibodies can be those described in Leslie Hudson and FrankC. Bay, Practical Immunology (2^(nd) Ed., Blackwell ScientificPublications 1980).

Animals (typically mice or rabbits) are injected with an JNK activatingphosphatase antigen and those with sufficient serum titer levels asdetermined by enzyme-linked immunosorbent assays (EIA) are selected forhybridoma production. Spleens of immunized animals are collected andprepared as single cell suspensions from which splenocytes arerecovered. The splenocytes are fused to mouse myeloma cells (such asSp2/0-Ag14 cells), allowed to incubate in Dulbeccos' Modified Eagle'Medium (DMEM) with 200 U/ml penicillin, 200 ug/ml streptomycin sulfate,and 4 mM glutamine, then incubated in HAT selection medium(hypoxanthine, aminopterin, and thymidine). After selection, tissueculture supernatants are taken from each fusion well and tested for JNKactivating phosphatase antibody production by EIA.

Alternative procedures for obtaining anti-JNK activating phosphataseantibodies may also be employed, such as immunization of transgenic miceharboring human Ig loci for production of fully human antibodies, andscreening of synthetic antibody libraries, such as those generated bymutagenesis of an antibody variable domain.

In this experiment, a peptide containing sequence of 5′ end of human JNKactivating phosphatase (SEQ ID NO: 22) was generated and used forantibody induction. This peptide was coupled with carrier proteinkeyhole limpet hemacyanin and injected into rabbits at a boost scheduleof once every two weeks. Titer of antibody against the peptide wasdetermined using ELISA assays with plates immobilized with the peptide.One of the animals (#1436) was determined to have the highest titeragainst the peptide. (H2N—CGNFKDARDAEQLS—COOH) (SEQ ID NO: 22)

EXAMPLE 10 Interaction of JKAP with HPK-1

Myc-tagged JKAP was coexpressed with HPK-1 in 293T cells. See FIG. 8(a).Lysates were prepared and HPK-1 was immunoprecipitated with ancommercially available anti-HPK-1 antibody (484). Co-immunoprecipitatedJKAP was detected with a commercially available anti-myc antibody.Equivalent levels of HPK-1 expression were confirmed by immunoblotanalysis using a commercially available anti-HPK-1 antibody (484). 293Tcells were co-transfected with 0.1 ug of HA-JNK1 alone, HA-JNK1 plus 2ug of either JKAP or HPK-1, or HA-JNK1 plus 2 ug each of both JKAP andHPK-1. See FIG. 8(b). Empty vectors were used to normalize the amount oftransfected DNA. Cell lysates were prepared, and HA-JNK1 wasimmunoprecipitated with an anti-HA antibody (12CA5). Immunocomplexkinase assays were performed using GST-cJune (1-79) as a substrate(WESTERN). 292T cells were transfected with 2 ug empty vector control,myc-JKAP, or myc-JKAP-C88S. Cell lysates were prepared andimmunoprecipitated with a commercially available anti-myc antibody. Theresulting precipitates were assayed for phosphatase activity byhydrolysis of p-nitrophenyl phosphate (pNPP). The enzyme reaction wasterminated after 30 minutes by addition of 3N NaOH and the productmeasured spectrophotometrically at 410 nm. See FIG. 8(c).

JKAP has been shown to have intrinsic phosphatase activity compared torelevant controls. The site-specific mutation of JKAP within an aminoacid residue that is conserved in all known dual-specificity phophatasesresults in complete abrogation of the observed phosphatase activity.JKAP could be specifically found to interact with HPK-1 aserine-theonine kinase that acts upstream of JNK in the JNK pathway. Inaddition, JKAP specifically synergizes with HPK-1 in the activation ofJNK. This result suggests that JKAP activates HPK-1. HPK-1 may be one ofthe natural substrates of JKAP although the precise residue that may bethe target for dephosphorylation is not discovered by these experiments.

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed.

1. An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) the nucleotide sequence asset forth in SEQ ID NOS: 1 or 3; (b) a nucleotide sequence encoding thepolypeptide as set forth in SEQ ID NOS: 2 or 4; (c) a nucleotidesequence corresponding to nucleotide position number 181 to 795 in SEQID NO: 1 or nucleotide position number 15 to 629 in SEQ ID NO:3; (d) anucleotide sequence encoding a polypeptide that is at least about 80,85, 90, 95, 96, 97, 98, or 99 percent identical to the nucleotidesequence of (c); (e) an allelic variant or splice variant of any of (a),(b, (c) or (d); (f) a nucleotide sequence of (b), (c), (d) or (e)encoding a polypeptide fragment of at least about 25, 50, 75, 100, orgreater than 100 amino acid residues wherein the polypeptide fragmenthas an activity of regulating JNK activation or modulating JNK-mediatedsignal transduction; (g) a nucleotide sequence of (a), (b), (c) or (d)comprising a fragment of at least about 10, 15, 20, 25, 50, 75, 100, orgreater than 100 nucleotides; (h) a nucleotide sequence encoding apolypeptide that has a substitution and/or deletion of 1 to 100 aminoacid residues as set forth in any of SEQ ID NOS: 2 or 4 wherein thepolypeptide has an activity of regulating JNK activation or modulatingJNK-mediated signal transduction; or serves as an antigen for generatingantibodies; and (i) a nucleotide sequence which hybridizes understringent conditions to any of (a)-(h); (j) a nucleotide sequencecomplementary to any of (a)-(h).
 2. An expression vector comprising thenucleic acid molecule of claim
 1. 3. A host cell comprising theexpression vector of claim
 2. 4. The host cell of claim 3 which is aeucaryotic cell.
 5. The host cell of claim 3 which is a prokaryoticcell.
 6. A process for producing a protein comprising growing a cultureof the host cell of claim 3 in suitable culture medium and isolating theprotein from the culture.
 7. A polypeptide produced by the process ofclaim
 6. 8. An isolated polypeptide comprising the amino acid sequenceselected from the group consisting of: (a) the amino acid sequence asset forth in SEQ ID NOS: 2 or 4; (b) a fragment of the amino acidsequence set forth in SEQ ID NOS: 2 or 4 comprising at least about 25,50, 75, 100, or greater than 100 amino acid residues wherein thefragment has an activity of regulating JNK activation or modulating.JNK-mediated signal transduction; or serves as an antigen for generatingantibodies; (c) an ortholog of SEQ ID NOS: 2 or 4; and (d) an allelicvariant or splice variant of (a), or (c).
 9. An isolated polypeptideencoded by the nucleic acid molecule of claim
 1. 10. An antibody orfragment thereof that specifically binds the polypeptide of claim
 8. 11.The antibody of claim 10 that is a monoclonal antibody.
 12. A fusionpolypeptide comprising the polypeptide of claim 8 fused to aheterologous amino acid sequence.
 13. The fusion polypeptide of claim 12wherein the heterologous amino acid sequence is an IgG constant domainor fragment thereof.
 14. A method of diagnosing a JNK-mediated disorderor a susceptibility to a JNK-mediated disorder in a subject comprising:(a) determining the presence or amount of expression of the polypeptideof claim 8 or the polypeptide encoded by the nucleic acid molecule ofclaim 1 in a sample; and (b) diagnosing a JNK-mediated disorder or asusceptibility to a JNK-mediated disorder based on the presence oramount of expression of the polypeptide.
 15. A method of identifying acompound which binds to a polypeptide comprising: (a) contacting thepolypeptide of claim 8 with a compound; and (b) determining the extentof binding of the polypeptide to the compound.
 16. A method ofmodulating levels of a polypeptide in an animal comprising administeringto the animal the nucleic acid molecule of claim
 1. 17. An antibodyproduced by immunizing an animal with a peptide comprising an amino acidsequence of SEQ ID NO:
 22. 18. A process for determining whether acompound inhibits JNK activating phosphatase polypeptide activitycomprising exposing a cell according to claim 3 to the compound, andmeasuring JNK activating phosphatase polypeptide activity in said cell.19. A method for producing the JNK-activating phosphatase comprising theamino acid sequence depicted in SEQ ID NOS: 2 or 4, said methodcomprising: (a) culturing the host cell of claim 3 under conditionssuitable for the expression of JNK-activating phosphatase; and (b)recovering JNK-activating phosphatase.
 20. An isolated JNK-activatingphosphatase protein having an amino-terminal dual-specificityphosphatase domain and lacking a noncatalytic carboxy-terminal domain;said protein having functional activities comprising dual-specificityphosphatase activity and JNK pathway upregulation activity.
 21. A methodfor screening a compound for the ability to augment JNK-activatingphosphatase activity in a host cell comprising (a) contacting saidcompound with a host cell of claim 3; and (b) determining the ability ofsaid compound to modulate the JNK-activating phosphatase activity ofsaid host cell, wherein a difference in said activity in the presence ofsaid compound is indicative of modulation activity by said compound.